Cyanine compounds

ABSTRACT

Compounds used as labels with properties comparable to known fluorescent compounds. The compounds can be conjugated to proteins and nucleic acids for biological imaging and analysis. Synthesis of the compounds, formation and use of the conjugated compounds, and specific non-limiting examples of each are provided.

This application is a continuation of U.S. application Ser. No.13/780,338 filed Feb. 28, 2013, which claims priority to U.S.application Ser. Nos. 61/606,210 filed Mar. 2, 2012 and 61/718,805 filedOct. 26, 2012, each of which is expressly incorporated by referenceherein in its entirety.

Compounds useful as labels with properties comparable to knownfluorescent compounds are disclosed. The compounds can be conjugated toproteins and nucleic acids for biological imaging and analysis.Synthesis of the compounds, formation and use of the conjugatedcompounds, and specific non-limiting examples of each are disclosed.

Compounds that react with biomolecules (e.g., antigens, antibodies,DNA-segments with the corresponding complimentary species for measuringenzyme kinetics, receptor-ligand interactions, nucleic acidhybridization kinetics in vitro as well as in vivo, etc.), termed labelsor dyes, are useful for, e.g., pharmacological characterization ofreceptors and drugs, binding data, etc. Compounds such as xanthyliumsalts (U.S. Pat. No. 5,846,737) and/or cyanines (U.S. Pat. No.5,627,027) are used for such applications, but aggregate and formdimers, especially in aqueous solution, due to planarity of theirπ-system. Compounds that have insufficient hydrophilicity undergonon-specific interactions with various surfaces, resulting in problemswhen attempting purify the corresponding conjugate, and anunsatisfactory signal to noise ratio.

Efforts are directed to reducing undesirable properties by introducingsubstituents that increase the hydrophilicity of the compounds. Forexample, sulfonic acid function substituents have been introduced intothe cyanine chromophore. U.S. Pat. No. 6,083,485 (Licha) and U.S. Pat.Nos. 6,977,305 and 6,974,873 (Molecular Probes) disclose cyaninecompounds having one of the common methyl groups in the 3-position ofthe terminal indole heterocycle substituted by a ω-carboxyalkylfunction, and in which the previously present (e.g. in Cy3 or Cy5)N-alkyl or N-ω-carboxyalkyl functions are replaced by N-w-alkyl sulfonicacid functions. WO 05/044923 discloses cyanine compounds having thecommon methyl substituent in the 3-position of the terminal indoleheterocycle substituted by a N-ω-alkyl sulfonic acid function. In thesepublications, cyanine compounds having more than two sulfonic acidfunction substituents exhibited higher solubility and correspondingly alower tendency to dimer formation, in comparison to cyanine compounds(Cy3, Cy5) described in U.S. Pat. No. 5,627,027.

The disclosed cyanine compounds are useful as labels in optical,especially fluorescence optical, determination and detection methods.The compounds have high hydrophilicity, high molar absorbance, highphoto-stability, and high storage stability. These compounds can beexcited by monochromatic (e.g., lasers, laser diodes) or polychromatic(e.g., white light sources) light in the ultraviolet (UV), visible, andnear infrared (NIR) spectral region to generate emission of fluorescencelight.

Typical application methods are based on the reaction of the compoundswith biomolecules such as proteins (e.g., antigens, antibodies, etc.),DNA and/or RNA segments, etc. with the corresponding complimentaryspecies. Thus, among other embodiments, the compounds are useful tomeasure enzyme kinetics, receptor-ligand interactions, and nucleic acidhybridization kinetics in vitro and/or in vivo. The compounds are usefulfor the pharmacological characterization of receptors and/or drugs.Applications include, but are not limited to, uses in medicine,pharmacy, biological sciences, materials sciences, environmentalcontrol, detection of organic and inorganic micro samples occurring innature, etc.

The following nomenclature is used to describe embodiments: 550 Compound1/X, 550 Compound 2/X, 550 Compound 3/X, 550 Compound 4/X, 550 Compound5/X, 550 Compound 6/X, 650 Compound 1/X, 650 Compound 2/X, 650 Compound3/X, 650 Compound 4/X, 650 Compound 5/X, 650 Compound 6/X, 755 Compound1/X, 755 Compound 2/X, 755 Compound 3/X, 755 Compound 4/X, 755 Compound5/X, 755 Compound 6/X, where 550, 650, and 755 Compounds comprise apolymethine chain of 3 carbon, 5 carbon, and 7 carbon atoms,respectively; the first number is the length of an ethylene glycol,diethylene glycol, or (poly)ethylene glycol (collectively referred toherein as PEG) on an indole N, e.g. 1 is ethylene glycol (PEG₁) on anindole N, 2 is diethylene glycol (PEG₂) on an indole N, 3 is(poly)ethylene glycol (poly=3, PEG₅) on an indole N, 4 is (poly)ethyleneglycol (poly=4, PEG₄) on an indole N, 5 is (poly)ethylene glycol(poly=5, PEG₅) on an indole N, and 6 is (poly)ethylene glycol (poly=6,PEG₆) on an indole N; and X is the total number of PEG groups on thecompound. For example, 650 Compound 4/4 contains PEG₄ on an indole N anda total of four PEG groups on the compound.

In one embodiment, the cyanine compounds have, in an N-position of oneheterocycle, ethylene glycol, diethylene glycol, or an ethylene glycolpolymer (i.e., poly(ethylene) glycol, abbreviated as PEG), and the otherheterocycle has, in a N-position, a function for conjugating thecompound to a biomolecule, and an ethylene glycol, diethylene glycol, oran ethylene glycol polymer (i.e., poly(ethylene) glycol, abbreviated asPEG) in another position of the cyanine compound. In one embodiment, thecyanine compound has, in any position of the compound, at least onesulfo and/or sulfoalkyl. In one embodiment, the cyanine compound has, inany position of the compound, a sulfonamide and/or carboxamidecomprising an ethylene glycol group or an ethylene glycol polymer (i.e.,poly(ethylene) glycol, abbreviated as PEG), either directly orindirectly attached to the compound. Indirect attachment indicates useof a linker, direct attachment indicates lack of such a linker. A linkercan be any moiety.

In one embodiment, the cyanine compounds have, in an N-position of oneheterocycle, an ethylene glycol, diethylene glycol, or ethylene glycolpolymer (i.e., poly(ethylene) glycol, abbreviated as PEG), and the otherheterocycle has, in a N-position, an ethylene glycol, diethylene glycol,or ethylene glycol polymer (i.e., poly(ethylene) glycol, abbreviated asPEG) and a function for conjugating the compound to a biomolecule, andan ethylene glycol group or an ethylene glycol polymer (i.e.,poly(ethylene) glycol, abbreviated as PEG) in another position of thebenzocyanine compound. In one embodiment, the cyanine compound has, inany position of the compound, at least one sulfo and/or sulfoalkyl. Inone embodiment, the cyanine compound has, in any position of thecompound, a sulfonamide and/or carboxamide comprising an ethyleneglycol, diethylene glycol, or ethylene glycol polymer (i.e.,poly(ethylene) glycol, abbreviated as PEG), either directly orindirectly attached to the compound. Indirect attachment indicates useof a linker, direct attachment indicates lack of such a linker. A linkercan be any moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the absorption maxima for compounds and conjugatedcompounds.

FIG. 2 shows fluorescence plate functional assay results with inventivecompounds and commercial dyes in one embodiment.

FIG. 3 shows fluorescence plate functional assay results with inventivecompounds and commercial dyes in one embodiment.

FIG. 4 shows fluorescence plate functional assay results with inventivecompounds and commercial dyes in one embodiment.

FIG. 5 shows fluorescence plate functional assay results with inventivecompounds and commercial dyes in one embodiment.

FIG. 6 shows fluorescence plate functional assay results with inventivecompounds and commercial dyes in one embodiment.

FIG. 7 shows fluorescence plate functional assay results with inventivecompounds and commercial dyes in one embodiment.

FIG. 8 shows fluorescence plate functional assay results with inventivecompounds and commercial dyes in one embodiment.

FIG. 9 shows fluorescence plate functional assay results with inventivecompounds and commercial dyes in one embodiment.

FIGS. 10A-B show the average total unbound fluorescence intensity withinventive compounds and commercial dyes in one embodiment.

FIGS. 11A-B show the average total unbound fluorescence intensity withinventive compounds and commercial dyes in one embodiment.

FIGS. 12A-B show the average total unbound fluorescence intensity withinventive compounds and commercial dyes in one embodiment.

FIGS. 13A-B show the average total unbound fluorescence intensity withinventive compounds and commercial dyes in one embodiment.

FIGS. 14A-E show immunofluorescence assay results with inventivecompounds and commercial dyes in one embodiment.

FIG. 15 shows the immunofluorescence assay results of FIG. 14 expressedas fluorescence intensity.

FIG. 16 shows the immunofluorescence assay results of FIG. 14 expressedas signal-to-background ratio.

FIGS. 17A-D show immunofluorescence assay results with inventivecompounds and commercial dyes in one embodiment.

FIG. 18 shows the immunofluorescence assay results of FIG. 17 expressedas fluorescence intensity.

FIG. 19 shows the immunofluorescence assay results of FIG. 17 expressedas signal-to-background ratio.

FIGS. 20A-D show immunofluorescence assay results with inventivecompounds and commercial dyes in one embodiment.

FIG. 21 shows the immunofluorescence assay results of FIG. 20 expressedas fluorescence intensity.

FIG. 22 shows the immunofluorescence assay results of FIG. 20 expressedas signal-to-background ratio.

Unless otherwise noted, reference to general formulas (e.g., I, II, III,IV, V, and VI, each subsequently described), encompasses theirrespective a, b, c, etc. structures.

In one embodiment, the compound is a compound according to generalformula Ia with “a” indicating the chain from the right indole Nterminates in COX:

or general formula Ib with “b” indicating the chain from the rightindole N terminates in OH:

where each of R¹ and R² is the same or different and is independentlyselected from the group consisting of an aliphatic, heteroaliphatic,sulfoalkyl, heteroaliphatic with terminal SO₃, a PEG group P-L-Z where Pis selected from an ethylene glycol group, a diethylene glycol group,and a (poly)ethylene glycol group where the (poly)ethylene glycol groupis (CH₂CH₂O)_(s) where s is an integer from 3-6 inclusive, a sulfonamidegroup -L-SO₂NH—P-L-Z, and a caboxamide group -L-CONH—P-L-Z; where L isselected from the group consisting of a divalent linear (—(CH₂)_(o)—,o=0 to 15), crossed, or cyclic alkane group that can be substituted byat least one atom selected from the group consisting of oxygen,substituted nitrogen, and/or sulfur; where Z is selected from the groupconsisting of H, CH₃, alkyl, heteroalkyl, NH₂, —COO⁻, —COOH, —COSH,CO—NH—NH₂, —COF, —COCl, —COBr, —COI, —COO—Su(succinimidyl/sulfo-succinimidyl), —COO—STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′—CO—CH₂—I, —CONR′R″, —CONR′-biomolecule, —CONR′-L-COO⁻,—CONR′-L-COOH, —CONR′-L-COO—Su, —CONR′-L-COO—STP, —CONR′-L-COO-TFP,—CONR′-L-CONR″₂, —CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂,—CONR′-L-OH, —CONR′-L-O-phosphoramidite, —CONR′-L-CHO,—CONR′-L-maleimide, and —CONR′-L-NH—CO—CH₂—I; R′ and R″ is selected fromthe group consisting of H, aliphatic group, and heteroaliphatic group,and the biomolecule is a protein, antibody, nucleotide, oligonucleotide,biotin, or hapten; X is selected from the group consisting of —OH, —SH,—NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHS(hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O—STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH2-I, where R is —H or an aliphatic or heteroaliphaticgroup; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive;and n is an integer from 1 to 3 inclusive; with the proviso that atleast one of R¹ and R² contains a PEG group.

In one embodiment, the PEG group is selected from —C—C—O—C (ethyleneglycol with terminal methyl), —C—C—O—C—C—O—C(diethylene glycol withterminal methyl), —C—C—O—C—C—O—C—C—O—C ((poly)ethylene glycol (3) withterminal methyl), —C—C—O—C—C—O—C—C—O—C—C—O—C((poly)ethylene glycol (4)with terminal methyl), —C—C—O—C—C—O—C—C—O—C—C—O—C—C—O—C ((poly)ethyleneglycol (5) with terminal methyl), or—C—C—O—C—C—O—C—C—O—C—C—O—C—C—C—O—C—C—O—C ((poly)ethylene glycol (6) withterminal methyl). In one embodiment, the PEG group P may be eitheruncapped, e.g., lack a terminal methyl, or may be capped with an atom orgroup other than a methyl. In one embodiment, the PEG group P terminateswith a Z group, where Z is selected from H, CH₃, a CH₃ group, alkyl, ora heteroalkyl group.

In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 1. In one embodiment thecompound is general formula I where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3; and nis 1. In one embodiment, the compound is general formula I where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; o is 3; and n is 1. In one embodiment thecompound is general formula I where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 1. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 1. In one embodiment thecompound is general formula I, where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and nis 1.

In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 2. In one embodiment thecompound is general formula I where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3; and nis 2. In one embodiment the compound is general formula I, where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; o is 3; and n is 2. In one embodiment thecompound is general formula I where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 2. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 2. In one embodiment thecompound is general formula I where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and nis 2.

In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 3. In one embodiment thecompound is general formula I where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3; and nis 3. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; o is 3; and n is 3. In one embodiment thecompound is general formula I where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 3. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 3. In one embodiment thecompound is general formula I where R1 is sulfoalkyl and R2 is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and nis 3.

In one embodiment the compound is general formula I where R1 is methyland R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; o is 3; and n is 1. In one embodiment the compound is general formulaI where R1 is methyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 1; o is 3; and n is 1. In one embodiment thecompound is general formula I where R1 is methyl and R2 is a PEG group;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 1.In one embodiment the compound is general formula I where R1 is methyland R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is3; o is 3; and n is 1. In one embodiment, the compound is generalformula I, where R1 is methyl and R2 is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 1. In oneembodiment the compound is general formula I where R1 is methyl and R2is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is3; and n is 1.

In one embodiment the compound is general formula I where R1 is methyland R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; o is 3; and n is 2. In one embodiment the compound is general formulaI where R1 is methyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 1; o is 3; and n is 2. In one embodiment thecompound is general formula I where R1 is methyl and R2 is a PEG group;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 2.In one embodiment the compound is general formula I where R1 is methyland R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is3; o is 3; and n is 2. In one embodiment the compound is general formulaI where R1 is methyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 2. In one embodiment thecompound is general formula I where R1 is methyl and R2 is a PEG group;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 2.

In one embodiment, the compound is general formula I where R1 is methyland R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is0; o is 3; and n is 3. In one embodiment, the compound is generalformula I where R1 is methyl and R2 is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 3. In oneembodiment the compound is general formula I where R1 is methyl and R2is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is3; and n is 3. In one embodiment, the compound is general formula Iwhere R1 is methyl and R2 is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; o is 3; and n is 3. In one embodiment, thecompound is general formula I where R1 is methyl and R2 is a PEG group;X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 3.In one embodiment, the compound is general formula I where R1 is methyland R2 is a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is5; o is 3; n is 3.

In one embodiment the compound is general formula I where R1 and R2 arePEG groups; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 0; o is 3;and n is 1. In one embodiment the compound is general formula I where R1and R2 are PEG groups; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is1; o is 3; and n is 1. In one embodiment the compound is general formulaI where R1 and R2 are PEG groups; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; o is 3; and n is 1. In one embodiment thecompound is general formula I where R1 and R2 are PEG groups; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and n is 1. In oneembodiment, the compound is general formula I, where R1 and R2 are PEGgroups; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; andn is 1. In one embodiment the compound is general formula I where R1 andR2 are PEG groups; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; ois 3; and n is 1.

In one embodiment the compound is general formula I where R1 and R2 arePEG groups; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 0; o is 3;and n is 2. In one embodiment the compound is general formula I where R1and R2 are PEG groups; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is1; o is 3; and n is 2. In one embodiment the compound is general formulaI where R1 and R2 are PEG groups; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; o is 3; and n is 2. In one embodiment thecompound is general formula I where R1 and R2 are PEG groups; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and n is 2. In oneembodiment the compound is general formula I where R1 and R2 are PEGgroups; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; andn is 2. In one embodiment the compound is general formula I where R1 andR2 are PEG groups; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; ois 3; and n is 2.

In one embodiment the compound is general formula I where R1 and R2 arePEG groups; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 0; o is 3;and n is 3. In one embodiment the compound is general formula I where R1and R2 are PEG groups; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is1; o is 3; and n is 3. In one embodiment the compound is general formulaI where R1 and R2 are PEG groups; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; o is 3; and n is 3. In one embodiment thecompound is general formula I where R1 and R2 are PEG groups; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and n is 3. In oneembodiment, the compound is general formula I, where R1 and R2 are PEGgroups; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; andn is 3. In one embodiment the compound is general formula I where R1 andR2 are PEG groups; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; ois 3; and n is 3.

In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 0; o is 3; and nis 1. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-′ maleimide; m is 1; o is 3; andn is 1. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and nis 1. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 1. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and nis 1. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and nis 1.

In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 0; o is 3; and nis 2. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3; and nis 2. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and nis 2. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 2. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and nis 2. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and nis 2.

In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 0; o is 3; and nis 3. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3; and nis 3. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 2; o is 3; and nis 3. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 3. In one embodiment, the compound is general formula I, where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; o is 3; and nis 3. In one embodiment the compound is general formula I where R1 issulfoalkyl and R2 is a sulfonamide group -L-SO₂NH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3; and nis 3.

In one embodiment, an isolated enantiomeric mixture selected fromdiastereomer Ia of general formula Ia shown below:

diastereomer Ib of general formula Ia shown below:

diastereomer Ic of general formula Ib shown below:

or diastereomer Id of general formula Ib shown below:

is provided, where each of R¹ and R² is the same or different and isindependently selected from the group consisting of aliphatic,heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO₃, a PEGgroup P-L-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a (poly)ethylene glycol group where the(poly)ethylene glycol group is (CH₂CH₂O)_(s) where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P-L-Z, and a caboxamidegroup -L-CONH—P-L-Z; where L is selected from the group consisting of adivalent linear (—(CH₂)_(o)—, o=0 to 15), crossed, or cyclic alkanegroup that can be substituted by at least one atom selected from thegroup consisting of oxygen, substituted nitrogen, and/or sulfur; where Zis selected from the group consisting of H, CH₃, alkyl, heteroalkyl,NH₂, —COO⁻, —COOH, —COSH, CO—NH—NH₂, —COF, —COCl, —COBr, —COI, —COO—Su(succinimidyl/sulfosuccinimidyl), —COO—STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′—CO—CH₂—I, —CONR′R″, —CONR′-biomolecule, —CONR′-L-COOH,—CONR′-L-COO—Su, —CONR′-L-COO—STP, —CONR′-L-COO-TFP, —CONR′-L-CONR″₂,—CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂, —CONR′-L-OH,—CONR′-L-O-phosphoramidite, —CONR′-L-CHO, —CONR′-L-maleimide, and—CONR′-L-NH—CO—CH₂—I; R′ and R″ is selected from the group consisting of—H, aliphatic, and heteroaliphatic, and the biomolecule is a protein,antibody, nucleotide, oligonucleotide, biotin, or hapten; X is selectedfrom the group consisting of —OH, —SH, —NH₂, —NH—NH₂, —F, —Cl, —Br, I,—NHS (hydroxysuccinimidyl/sulfo-succinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O—STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH2-I, where R is —H or an aliphatic or heteroaliphaticgroup; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive;and n is an integer from 1 to 3 inclusive; with the proviso that atleast one of R¹ and R² contains a PEG group.

In one embodiment, the compound has general formula IIa with “a”indicating the chain from the right indole N terminates in COX:

or general formula IIb with “b” indicating the chain from the rightindole N terminates in COH:

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of aliphatic,heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO₃, a PEGgroup P-L-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a (poly)ethylene glycol group where the(poly)ethylene glycol group is (CH₂CH₂O)_(s) where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P-L-Z, and a caboxamidegroup -L-CONH—P-L-Z; each of R⁷ and R⁸ is the same or different and isindependently selected from the group consisting of H, SO₃, a PEG groupP-L-Z where P is selected from an ethylene glycol group, a diethyleneglycol group, and a (poly)ethylene glycol group where the (poly)ethyleneglycol group is (CH₂CH₂O)_(s) where s is an integer from 3-6 inclusive,a sulfonamide group —SO₂NH—P-L-Z, and a caboxamide group —CONH—P-L-Z;where L is selected from the group consisting of a divalent linear(—(CH₂)_(o)—, o=0 to 15), crossed, or cyclic alkane group that can besubstituted by at least one atom selected from the group consisting ofoxygen, substituted nitrogen, and/or sulfur; where Z is selected fromthe group consisting of H, CH₃, alkyl, heteroalkyl, NH₂, —COO⁻, —COOH,—COSH, CO—NH—NH₂, —COF, —COCl, —COBr, —COI, —COO—Su(succinimidyl/sulfosuccinimidyl), —COO—STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′—CO—CH₂—I, —CONR′R″, —CONR′-biomolecule, —CONR′-L-COO⁻,—CONR′-L-COOH, —CONR′-L-COO—Su, —CONR′-L-COO—STP, —CONR′-L-COO-TFP,—CONR′-L-CONR″₂, —CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂,—CONR′-L-OH, —CONR′-L-O-phosphoramidite, —CONR′-L-CHO,—CONR′-L-maleimide, and —CONR′-L-NH—CO—CH₂—I; R′ and R″ is selected fromthe group consisting of H, aliphatic group, and heteroaliphatic group,and the biomolecule is a protein, antibody, nucleotide, oligonucleotide,biotin, or hapten; X is selected from the group consisting of —OH, —SH,—NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHS(hydroxysuccinimidyl/sulfo-succinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O—STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH₂—I, where R is —H or an aliphatic or heteroaliphaticgroup; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive;and n is an integer from 1 to 3 inclusive; with the proviso that atleast one of R¹, R², R⁵, R⁶, R⁷, and R⁸ contains a PEG group.

In one embodiment the compound is general formula II where R1, R5, andR6 are methyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 0; o is 3; and n is 1. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 1. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 1. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; o is 3; and n is 1. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 1. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 1.

In one embodiment the compound is general formula II where R1, R5, andR6 are methyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 0; o is 3; and n is 2. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 2. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 2. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; o is 3; and n is 2. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 2. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 2.

In one embodiment the compound is general formula II where R1, R5, andR6 are methyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 0; o is 3; and n is 3. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 3. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 3. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; o is 3; and n is 3. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 3. In oneembodiment the compound is general formula II where R1, R5, and R6 aremethyl and R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 3.

In one embodiment the compound is general formula II where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 0; o is 3; and n is 1. In oneembodiment the compound is general formula II where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 1. In oneembodiment the compound is general formula II where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 1. In oneembodiment the compound is general formula II where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; o is 3; and n is 1. In oneembodiment, the compound is general formula II, where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 1. In oneembodiment the compound is general formula II where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 1.

In one embodiment the compound is general formula II where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 0; o is 3; and n is 2. In oneembodiment the compound is general formula II where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 2. In oneembodiment the compound is general formula II, where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 2. In oneembodiment, the compound is general formula II, where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; o is 3; and n is 2. In oneembodiment, the compound is general formula II, where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 2. In oneembodiment, the compound is general formula II, where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 2.

In one embodiment the compound is general formula II where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 0; o is 3; and n is 3. In oneembodiment the compound is general formula II where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; o is 3; and n is 3. In oneembodiment the compound is general formula II where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; o is 3; and n is 3. In oneembodiment the compound is general formula II where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; o is 3; and n is 3. In oneembodiment, the compound is general formula II, where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; o is 3; and n is 3. In oneembodiment the compound is general formula II where R5 and R6 aremethyl; R1 and R2 are PEG groups; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; o is 3; and n is 3.

In one embodiment, the compound is general formula II, where R5 and R6are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 is sulfonamide-L-SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is sulfonamide -L-SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3;and n is 1. In one embodiment, the compound is general formula II, whereR5 and R6 are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 issulfonamide -L-SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 2; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is sulfonamide -L-SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3;and n is 1. In one embodiment, the compound is general formula II, whereR5 and R6 are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 issulfonamide -L-SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 4; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is sulfonamide -L-SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3;and n is 1.

In one embodiment, the compound is general formula II, where R5 and R6are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 is sulfonamide-L-SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is sulfonamide -L-SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3;and n is 2. In one embodiment, the compound is general formula II, whereR5 and R6 are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 issulfonamide -L-SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 2; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is sulfonamide -L-SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3;and n is 2. In one embodiment, the compound is general formula II, whereR5 and R6 are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 issulfonamide -L-SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 4; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is sulfonamide -L-SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3;and n is 2.

In one embodiment, the compound is general formula II, where R5 and R6are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 is sulfonamide-L-SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is sulfonamide -L-SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3;and n is 3. In one embodiment, the compound is general formula II, whereR5 and R6 are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 issulfonamide -L-SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 2; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is sulfonamide -L-SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3;and n is 3. In one embodiment, the compound is general formula II, whereR5 and R6 are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 issulfonamide -L-SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 4; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is sulfonamide -L-SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3;and n is 3.

In one embodiment, the compound is general formula II, where R5 and R6are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 is a caboxamidegroup —CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is a caboxamide group —CONH—P where P isa PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3;and n is 1. In one embodiment, the compound is general formula II, whereR5 and R6 are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 is acaboxamide group —CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 2; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is a caboxamide group —CONH—P where P isa PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3;and n is 1. In one embodiment, the compound is general formula II, whereR5 and R6 are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 iscaboxamide —CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 1. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is a caboxamide group —CONH—P where P isa PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3;and n is 1.

In one embodiment, the compound is general formula II, where R5 and R6are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 is a caboxamidegroup —CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is a caboxamide —CONH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3; and nis 2. In one embodiment, the compound is general formula II, where R5and R6 are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 iscaboxamide —CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is caboxamide —CONH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 2. In one embodiment, the compound is general formula II, where R5and R6 are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 iscaboxamide —CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 2. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is a caboxamide group —CONH—P where P isa PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3;and n is 2.

In one embodiment, the compound is general formula II, where R5 and R6are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 is a caboxamidegroup —CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is a caboxamide group —CONH—P where P isa PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; o is 3;and n is 3. In one embodiment, the compound is general formula II, whereR5 and R6 are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 iscaboxamide —CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 2; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is carboxamide —CONH—P where P is a PEGgroup; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; o is 3; and nis 3. In one embodiment, the compound is general formula II, where R5and R6 are methyl; R1 and R2 are PEG groups; R7 is sulfo; R8 iscaboxamide —CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 4; o is 3; and n is 3. In one embodiment, thecompound is general formula II, where R5 and R6 are methyl; R1 and R2are PEG groups; R7 is sulfo; R8 is a caboxamide group —CONH—P where P isa PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; o is 3;and n is 3.

In one embodiment, the compound has general formula IIIa with “a”indicating the chain from the right indole N terminates in COX:

or general formula IIIb with “b” indicating the chain from the rightindole N terminates in COH:

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P-L-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a (poly)ethylene glycol group where the(poly)ethylene glycol group is (CH₂CH₂O)_(s) where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P-L-Z, and a caboxamidegroup -L-CONH—P-L-Z; each of R⁷ and R⁸ is the same or different and isindependently selected from the group consisting of H, SO₃, a PEG groupP-L-Z where P is selected from an ethylene glycol group, a diethyleneglycol group, and a (poly)ethylene glycol group where the (poly)ethyleneglycol group is (CH₂CH₂O)_(s) where s is an integer from 3-6 inclusive,a sulfonamide group —SO₂NH—P-L-Z, and a caboxamide group —CONH—P-L-Z;where L is selected from the group consisting of a divalent linear(—(CH₂)_(o)—, o=0 to 15), crossed, or cyclic alkane group that can besubstituted by at least one atom selected from the group consisting ofoxygen, substituted nitrogen, and/or sulfur; where Z is selected fromthe group consisting of H, CH₃, alkyl, heteroalkyl, NH₂, —COO⁻, —COOH,—COSH, CO—NH—NH₂, —COF, —COCl, —COBr, —COI, —COO—Su(succinimidyl/sulfosuccinimidyl), —COO—STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′R″, —CONR′-biomolecule, —CONR′-L-COOH, —CONR′-L-COO—Su,—CONR′-L-COO—STP, —CONR′-L-COO-TFP, —CONR-L-CONR″₂,—CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂, —CONR′-L-OH,—CONR′-L-O-phosphoramidite, —CONR′-L-CHO, —CONR′-L-maleimide, and—CONR′-L-NH—CO—CH₂—I; R′ and R″ is selected from the group consisting ofH, aliphatic group, and heteroaliphatic group, and the biomolecule is aprotein, antibody, nucleotide, oligonucleotide, biotin, or hapten; X isselected from the group consisting of —OH, —SH, —NH₂, —NH—NH₂, —F, —Cl,—Br, I, —NHS (hydroxysuccinimidyl/sulfo-succinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O—STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH2-I, where R is —H or an aliphatic or heteroaliphaticgroup; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; p is an integer from 1 to 6 inclusive;and n is an integer from 1 to 3 inclusive; and at least one of R¹, R²,R⁵, R⁶, R⁷, and R⁸ contains a PEG group.

In one embodiment, the compound is general formula III, where R1, R5,and R6 are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 0; p is 1; and n is 1. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; p is 2; and n is 1. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 1. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; p is 4; and n is 1. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; p is 5; and n is 1. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 1.

In one embodiment, the compound is general formula III, where R1, R5,and R6 are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 0; p is 1; and n is 2. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; p is 2; and n is 2. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 2. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; p is 4; and n is 2. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; p is 5; and n is 2. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 2.

In one embodiment, the compound is general formula III, where R1, R5,and R6 are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 0; p is 1; and n is 3. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; p is 2; and n is 3. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 3. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; p is 4; and n is 3. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; p is 5; and n is 3. In oneembodiment, the compound is general formula III, where R1, R5, and R6are methyl; R2 is a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 3.

In one embodiment the compound is general formula III where R5 and R6are methyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 0; p is 1; and n is 1. In oneembodiment the compound is general formula III where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; p is 2; and n is 1. In oneembodiment the compound is general formula III where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 1. In oneembodiment the compound is general formula III where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; p is 4; and n is 1. In oneembodiment the compound is general formula III where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; p is 5; n is 1. In one embodimentthe compound is general formula III where R5 and R6 are methyl; R1 andR2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 5; p is 6; n is 1.

In one embodiment, the compound is general formula III, where R5 and R6are methyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 0; p is 1; and n is 2. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; p is 2; and n is 2. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 2. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; p is 4; and n is 2. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; p is 5; and n is 2. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 2.

In one embodiment, the compound is general formula III, where R5 and R6are methyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH,—NHS, —O-TFP, or —NR-L-maleimide; m is 0; p is 1; and n is 3. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 1; p is 2; and n is 3. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 3. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 3; p is 4; and n is 3. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 4; p is 5; and n is 3. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 and R8 are sulfo; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 3.

In one embodiment, the compound is general formula III, where R5 and R6are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8 is sulfonamide—SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 1. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is sulfonamide —SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is 2;and n is 1. In one embodiment, the compound is general formula III,where R5 and R6 are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8is sulfonamide —SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 2; p is 3; and n is 1. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is sulfonamide —SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; p is 4;and n is 1. In one embodiment, the compound is general formula III,where R5 and R6 are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8is sulfonamide —SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 4; p is 5; and n is 1. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is sulfonamide —SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; p is 6;and n is 1.

In one embodiment, the compound is general formula III, where R5 and R6are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8 is sulfonamide—SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 2. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is sulfonamide —SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is 2;and n is 2. In one embodiment, the compound is general formula III,where R5 and R6 are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8is sulfonamide —SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 2; p is 3; and n is 2. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is sulfonamide —SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; p is 4;and n is 2. In one embodiment, the compound is general formula III,where R5 and R6 are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8is sulfonamide —SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 4; p is 5; and n is 2. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is sulfonamide —SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; p is 6;and n is 2.

In one embodiment, the compound is general formula III, where R5 and R6are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8 is sulfonamide—SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 3. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is sulfonamide —SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is 2;and n is 3. In one embodiment, the compound is general formula III,where R5 and R6 are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8is sulfonamide —SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 2; p is 3; and n is 3. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is sulfonamide —SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 3; p is 4;and n is 3. In one embodiment, the compound is general formula III,where R5 and R6 are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8is sulfonamide —SO₂NH—P where P is a PEG group; X is —OH, —NHS, —O-TFP,or —NR-L-maleimide; m is 4; p is 5; and n is 3. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is sulfonamide —SO₂NH—P where P is aPEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 5; p is 6;and n is 3.

In one embodiment, the compound is general formula III, where R5 and R6are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8 is a caboxamidegroup —CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 1. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is a caboxamide group —CONH—P where Pis a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 1. In one embodiment, the compound is general formula III,where R5 and R6 are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8is a caboxamide group —CONH—P where P is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 1. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 is sulfo; R8 is a caboxamide group—CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 1. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is a caboxamide group —CONH—P where Pis a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 1. In one embodiment, the compound is general formula III,where R5 and R6 are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8is a caboxamide group —CONH—P where P is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 1.

In one embodiment, the compound is general formula III, where R5 and R6are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8 is a caboxamidegroup —CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 2. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is a caboxamide group —CONH—P where Pis a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 2. In one embodiment, the compound is general formula III,where R5 and R6 are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8is a caboxamide group —CONH—P where P is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 2. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 is sulfo; R8 is a caboxamide group—CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 2. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is a caboxamide group —CONH—P where Pis a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 2. In one embodiment, the compound is general formula III,where R5 and R6 are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8is a caboxamide group —CONH—P where P is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 2.

In one embodiment, the compound is general formula III, where R5 and R6are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8 is a caboxamidegroup —CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 0; p is 1; and n is 3. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is a caboxamide group —CONH—P where Pis a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 1; p is2; and n is 3. In one embodiment, the compound is general formula III,where R5 and R6 are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8is a caboxamide group —CONH—P where P is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 2; p is 3; and n is 3. In oneembodiment, the compound is general formula III, where R5 and R6 aremethyl; R1 and R2 are a PEG group; R7 is sulfo; R8 is a caboxamide group—CONH—P where P is a PEG group; X is —OH, —NHS, —O-TFP, or—NR-L-maleimide; m is 3; p is 4; and n is 3. In one embodiment, thecompound is general formula III, where R5 and R6 are methyl; R1 and R2are a PEG group; R7 is sulfo; R8 is a caboxamide group —CONH—P where Pis a PEG group; X is —OH, —NHS, —O-TFP, or —NR-L-maleimide; m is 4; p is5; and n is 3. In one embodiment, the compound is general formula III,where R5 and R6 are methyl; R1 and R2 are a PEG group; R7 is sulfo; R8is a caboxamide group —CONH—P where P is a PEG group; X is —OH, —NHS,—O-TFP, or —NR-L-maleimide; m is 5; p is 6; and n is 3.

In one embodiment, the compound is 550 Compound 1/2

550 Compound 1/2(1-(5-carboxypentyl)-3-(2-methoxyethyl)-2-((1E,3E)-3-(1-(2-methoxyethyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)prop-1-enyl)-3-methyl-3H-indolium-5-sulfonate)contains an ethylene glycol on the indole N of the left heterocycle,i.e., a methylated ethylene glycol, as shown in the structure above, andthe ethylene glycol can be represented in abbreviated format as—[C—C—O]₁—, which is used throughout. The methyl group on the ethyleneglycol prevents the terminal —OH from oxidation. Oxidation is known tooccur, over time, on an unprotected terminus of an ethylene glycolgroup, diethylene glycol group, or (poly)ethylene glycol group,collectively referred to herein as an unprotected PEG terminus. Adding amethyl ether provides this protection, and prevents reaction withelectrophilic reactive groups.

In embodiments, e.g., for functional assays, the inventive compounds areactivated. Activation of the compound adds a chemical moiety such thatthe compound is in a form that can be conjugated to a biological moiety.Examples of chemical moieties for activation are described below withreference to activation of 550 Compound 1, but one skilled in the artappreciates that activation is not limited to these examples. Onenon-limiting example of an activated compound is the NHS-ester of 550Compound 1/2, shown below:

In one embodiment, the compound is a NHS-ester of 550 Compound 1/2where, according to general formula I, o is 1, shown below:

In one embodiment, the compound is an NHS-ester of 550 Compound 1/2where, according to general formula I, o is 5, shown below:

One non-limiting example of a NHS-ester of 550 Compound 1/3, accordingto general formula III, where m=1 and p=1, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 1/3, accordingto general formula III, where m=1 and p=2, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 1/3, accordingto general formula III, where m=1 and p=3, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 1/3, accordingto general formula III, where m=1 and p=4, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 1/3, accordingto general formula III, where m=1 and p=5, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 1/3, accordingto general formula III, where m=1 and p=6, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 2/3, accordingto general formula III, where m=2 and p=1, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 2/3, accordingto general formula III, where m=2 and p=2, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 2/3, accordingto general formula III, where m=2 and p=3, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 3/3, accordingto general formula III, where m=3 and p=1, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 3/3, accordingto general formula III, where m=3 and p=2, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 3/3, accordingto general formula III, where m=3 and p=3, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 4/3, accordingto general formula III, where m=4 and p=1, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 5/3, accordingto general formula III, where m=5 and p=1, is shown below:

One non-limiting example of a NHS-ester of 550 Compound 6/3, accordingto general formula III, where m=6 and p=1, is shown below:

One non-limiting example of an activated 550 Compound 1/2 is atetrafluorophenyl (TFP)-ester form of 550 Compound 1, shown below:

One non-limiting example of an activated 550 Compound 1/2 is asulfotetrafluorophenyl (STP)-ester form of 550 Compound 1, shown below:

One non-limiting example of an activated 550 Compound 1/2 is a hydrazideform of 550 Compound 1, shown below:

One non-limiting example of an activated 550 Compound 1/2 is a maleimideform of 550 Compound 1,

In one embodiment, the compound is 550 Compound 2/2

550 Compound 2/2(1-(5-carboxypentyl)-2-((1E,3E)-3-(1-(2-(2-methoxyethoxy)ethyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)prop-1-enyl)-3-(2-methoxyethyl)-3-methyl-3H-indolium-5-sulfonate)contains a diethylene glycol on the indole N of the left heterocycle.550 Compound 2/2, with the diethylene glycol shown in abbreviatednotation used throughout, represents the following structure.

The methyl group on the ethylene glycol prevents the terminal —OH fromoxidation. Oxidation is known to occur, over time, on an unprotected PEGterminus. Adding a methyl ether provides this protection, and preventsreaction with electrophilic reactive groups. For functional assays, 550Compound 2/2 is activated as described above, one non-limiting exampleof which is the NHS-ester form of 550 Compound 2/2, shown below.

In one embodiment, the compound is 550 Compound 3/2

550 Compound 3/2(1-(5-carboxypentyl)-2-((1E,3E)-3-(1-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)prop-1-enyl)-3-(2-methoxyethyl)-3-methyl-3H-indolium-5-sulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. 550 Compound 3/2, with the (poly)ethylene glycol shown inabbreviated notation used throughout, represents the followingstructure.

The methyl group on the ethylene glycol prevents the terminal —OH fromoxidation. Oxidation is known to occur, over time, on an unprotected PEGterminus. Adding a methyl ether provides this protection, and preventsreaction with electrophilic reactive groups. For functional assays, 550Compound 3/2 is activated as described above.

In one embodiment, the compound is 550 Compound 4/2

550 Compound 4/2(1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methyl-2-((1E,3E)-3-(3-methyl-5-sulfonato-3-(3-sulfonatopropyl)-1-(2,5,8,11-tetraoxatridecan-13-yl)indolin-2-ylidene)prop-1-enyl)-3H-indolium-5-sulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. 550 Compound 4/2, with the (poly)ethylene glycol shown inabbreviated notation used throughout, represents the followingstructure.

The methyl group on the ethylene glycol prevents the terminal —OH fromoxidation. Oxidation is known to occur, over time, on an unprotected PEGterminus. Adding a methyl ether provides this protection, and preventsreaction with electrophilic reactive groups. For functional assays, 550Compound 4/2 is activated as described above.

In one embodiment, the compound is 550 Compound 5/2

550 Compound 5/2(2-((1E,3E)-3-(1-(2,5,8,11,14-pentaoxahexadecan-16-yl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)prop-1-enyl)-1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methyl-3H-indolium-5-sulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. 550 Compound 5/2, with the (poly)ethylene glycol shown inabbreviated notation used throughout, represents the followingstructure.

The methyl group on the ethylene glycol prevents the terminal —OH fromoxidation. Oxidation is known to occur, over time, on an unprotected PEGterminus. Adding a methyl ether provides this protection, and preventsreaction with electrophilic reactive groups. For functional assays, 550Compound 5/2 is activated as described above.

In one embodiment, the compound is 550 Compound 6/2

550 Compound 6/2(1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methyl-2-((1E,3E)-3-(3-methyl-1-(2,5,8,11,14,17-hexaoxanonadecan-19-yl)-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)prop-1-enyl)-3H-indolium-5-sulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. 550 Compound 6/2, with the (poly)ethylene glycol shown inabbreviated notation used throughout, represents the followingstructure.

The methyl group on the ethylene glycol prevents the terminal —OH fromoxidation. Oxidation is known to occur, over time, on an unprotected PEGterminus. Adding a methyl ether provides this protection, and preventsreaction with electrophilic reactive groups. For functional assays, 550Compound 6/2 is activated as described above.

In embodiments, the degree of sulfonation is varied to, e.g., vary thecompound's degree of hydrophilicity or hydrophobicity. One non-limitingexample is a monosulfonate form of 550 Compound 1/2, shown below, but itis understood that the single sulfo group can be at any of the describedpositions:

One non-limiting example is a disulfonate form of 550 Compound 1/2,shown below, but it is understood that each of the two sulfo groups canbe at any of the described positions:

One non-limiting example is a trisulfonate form of 550 Compound 1/2,shown below, but it is understood that each of the three sulfo groupscan be at any of the described positions:

One non-limiting example is a tetrasulfonate form of 550 Compound 1/2,shown below, but it is understood that each of the four sulfo groups canbe at any of the described positions:

In embodiments, the compound contains one or more substitutions of thepolymethine linker. In one embodiment, the compound has general formulaIVa with “a” indicating an ethylene glycol, diethylene glycol, or(poly)ethylene glycol group on the left indole N, and the chain on theright indole N terminating in COX:

general formula IVb with “b” indicating an ethylene glycol, diethyleneglycol, or (poly)ethylene glycol group on the left indole N, and thechain on the right indole N terminating in COH:

general formula IVc with “c” indicating an ethylene glycol, diethyleneglycol, or (poly)ethylene glycol group on the left and right indole N,and the chain on the right indole N terminating in COX:

or general formula IVd with “d” indicating an ethylene glycol,diethylene glycol, or (poly)ethylene glycol group on the left indole N,and the chain on the right indole N terminating in COH:

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO₃, a PEGgroup P-L-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a (poly)ethylene glycol group where the(poly)ethylene glycol group is (CH₂CH₂O)_(s) where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P-L-Z, and a caboxamidegroup -L-CONH—P-L-Z; each of R⁷ and R⁸ is the same or different and isindependently selected from either H, SO₃, a PEG group P-L-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and a(poly)ethylene glycol group, where the (poly)ethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup —SO₂NH—P-L-Z, or a caboxamide group —CONH—P-L-Z; where L isselected from the group consisting of a divalent linear (—(CH₂)_(s)—,o=0 to 15), crossed, or cyclic alkane group that can be substituted byat least one atom selected from the group consisting of oxygen,substituted nitrogen, and/or sulfur; where Z is selected from the groupconsisting of H, CH₃, alkyl, a heteroalkyl group, NH₂, —COO⁻, —COOH,—COSH, CO—NH—NH₂, —COF, —COCl, —COBr, —COI, —COO—Su(succinimidyl/sulfo-succinimidyl), —COO-STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′—CO—CH₂—I, —CONR′R″, —CONR′-biomolecule, —CONR′-L-COO⁻,—CONR′-L-COOH, —CONR′-L-COO—Su, —CONR′-L-COO—STP, —CONR′-L-COO-TFP,—CONR′-L-CONR″₂, —CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂,—CONR′-L-OH, —CONR′-L-O-phosphoramidite, —CONR′-L-CHO,—CONR′-L-maleimide, and —CONR′-L-NH—CO—CH₂—I; R′ and R″ is selected fromthe group consisting of H, aliphatic group, and heteroaliphatic group,and the biomolecule is a protein, antibody, nucleotide, oligonucleotide,biotin, or hapten; X is selected from the group consisting of —OH, —SH,—NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHShydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O—STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH2-1, where R is —H or an aliphatic or heteroaliphaticgroup; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; p is an integer from 1 to 6 inclusive;each of R3 and R4 is the same or different and is independentlyhydrogen, an aliphatic group, a heteroaliphatic group, or a PEG groupP-L-Z where P is selected from an ethylene glycol group, a diethyleneglycol group, and a (poly)ethylene glycol group where the (poly)ethyleneglycol group is (CH₂CH₂O)_(s) where s is an integer from 3-6 inclusive;or R3 and R4 together form a cyclic structure where R3 and R4 are joinedusing a divalent structural element selected from the group consistingof —(CH₂)_(q)—, —(CH₂)_(q)O(CH₂)_(q′), —(CH₂)_(q)S(CH₂)_(q′)—,—(CH₂)_(q)CH═CH—, —OCH═CH— where each of q and q′ is the same ordifferent and is a integer from 2 to 6 inclusive; and Y is selected fromthe group consisting of hydrogen, alkyl, sulfoalkyl, fluorine, chlorine,bromine, a substituted or unsubstituted aryl-, phenoxy- orphenylmercapto function; and Y is selected from the group consisting ofhydrogen, alkyl, sulfoalkyl, fluorine, chlorine, bromine, and a PEGgroup P-L-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a (poly)ethylene glycol group where the(poly)ethylene glycol group is (CH₂CH₂O)_(s) where s is an integer from3-6 inclusive; with the proviso that at least one of R¹, R², R³, R⁵, R⁶,R⁷, and R⁸ contains a PEG group.

In one embodiment, the compound of general formula IV wherein each of R3and R4 is the same or different and is independently hydrogen, analiphatic group, or a heteroaliphatic group, or R3 and R4 together forma cyclic structure where R3 and R4 are directly joined or joined using adivalent structural element selected from the group consisting of—(CH₂)_(q)— and CH═CH, where q is an integer from 1 to 2 inclusive, toresult in a 3-, 4-, or 5-membered ring.

One non-limiting example is a substituted polymethine form of 550Compound 1/2, shown below:

One non-limiting example is a substituted polymethine form of 550Compound 2/2, shown below:

One non-limiting example is a substituted polymethine form of 550Compound 3/2, shown below:

One non-limiting example is a substituted polymethine form of 550Compound 4/2, shown below:

One non-limiting example is a substituted polymethine form of 550Compound 5/2, shown below:

One non-limiting example is a substituted polymethine form of 550Compound 6/2, shown below:

One non-limiting example is a substituted polymethine form of 550 havingan ethylene glycol, diethylene glycol, or (poly)ethylene glycol asdescribed for general formula IV, such as the compound shown below:

In embodiments, an ethylene glycol group, diethylene glycol group,and/or a (poly)ethylene glycol group, which will collectively bereferred to as a PEG group, unless specifically defined, may be presentat position(s) in addition to such groups being present on the indole Natom(s).

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R1 is anethylene glycol group terminating with a methyl group,

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R1 is adiethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R1 is a(poly)ethylene glycol (3) group terminating with a methyl

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R1 is a(poly)ethylene glycol (4) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R1 is a(poly)ethylene glycol (5) group terminating with a methyl

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R1 is a(poly)ethylene glycol (6) group terminating with a methyl

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R1 is asulfonamide group -L-SO₂NH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R1 is acarboxamide group -L-CONH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R2 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R2 is adiethylene glycol group terminating with a methyl group,

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R2 is a(poly)ethylene glycol (3) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R2 is a(poly)ethylene glycol (4) group terminating with a methyl

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R2 is a(poly)ethylene glycol (5) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R2 is a(poly)ethylene glycol (6) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R2 is asulfonamide group -L-SO₂NH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R2 is acarboxamide group -L-CONH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/3 according to general formula II where both R1 and R2are an ethylene glycol group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/3 according to general formula II where both R1 and R2are a diethylene glycol group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/3 according to general formula II where both R1 and R2are a (poly)ethylene glycol (3) group terminating with a methyl group,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/3 according to general formula II where both R1 and R2are a (poly)ethylene glycol (4) group terminating with a methyl group,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 4/4 according to general formula III where both R1 and R2are a (poly)ethylene glycol (4) group terminating with a methyl group,and R7 and R8 are sulfo, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 4/4 according to general formula III where both R1 and R2are a (poly)ethylene glycol (4) group terminating with a methyl group,and R7 and R8 are H, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/3 according to general formula II where both R1 and R2are a (poly)ethylene glycol (5) group terminating with a methyl group,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/3 according to general formula II where both R1 and R2are a (poly)ethylene glycol (6) group terminating with a methyl group,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R8 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R8 issulfonamide —SO₂NH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R8 iscarboxamide —CONH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R7 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R7 is asulfonamide group —SO₂NH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/2 according to general formula II where R7 is acarboxamide group —CONH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/3 according to general formula II where both R7 and R8are an ethylene glycol group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/3 according to general formula II where both R7 and R8are a sulfonamide group —SO₂NH—P—Z where Z is a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 550 Compound 1/3 according to general formula II where both R7 and R8are a carboxamide group —CONH—P—Z where Z is a methyl group, shownbelow:

In one embodiment, the compound is 650 Compound 1/2

650 Compound 1/2((2-((1E,3E,5E)-5-(1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methyl-5-sulfonatoindolin-2-ylidene)penta-1,3-dienyl)-1-(2-methoxyethyl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonatetri sodium salt) contains an ethylene glycol on the indole N of the leftheterocycle, i.e., a methylated ethylene glycol. The methyl group on theethylene glycol prevents the terminal —OH from oxidation. Oxidation isknown to occur, over time, on an unprotected PEG terminus (i.e., anunprotected ethylene glycol group, diethylene glycol group, or(poly)ethylene glycol group). Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.

In embodiments, e.g., for functional assays, the inventive compounds areactivated. Activation of the compound adds a chemical moiety such thatthe compound is in a form that can be conjugated to a biological moiety.Examples of chemical moieties for activation are described below withreference to activation of 650 Compound 1, but one skilled in the artappreciates that activation is not limited to these examples. Onenon-limiting example of an activated compound is the NHS-ester of 650Compound 1/2,

In one embodiment, the compound is a NHS-ester of 650 Compound 1/2where, according to general formula I, o is 1, shown below:

In one embodiment, the compound is an NHS-ester of 650 Compound 1/2where, according to general formula I, o is 5, shown below:

One non-limiting example of a NHS-ester of 650 Compound 1/3, accordingto formula III, where m=1 and p=1, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 1/3, accordingto general formula III, where m=1 and p=2, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 1/3, accordingto general formula III, where m=1 and p=3, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 1/3, accordingto general formula III, where m=1 and p=4, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 1/3, accordingto general formula III, where m=1 and p=5, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 1/3, accordingto general formula III, where m=1 and p=6, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 2/3, accordingto general formula III, where m=2 and p=1, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 2/3, accordingto general formula III, where m=2 and p=2, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 2/3, accordingto general formula III, where m=2 and p=3, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 3/3, accordingto general formula III, where m=3 and p=1, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 3/3, accordingto general formula III, where m=3 and p=2, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 3/3, accordingto general formula III, where m=3 and p=3, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 4/3, accordingto general formula III, where m=4 and p=1, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 5/3, accordingto general formula III, where m=5 and p=1, is shown below:

One non-limiting example of a NHS-ester of 650 Compound 6/3, accordingto general formula III, where m=6 and p=1, is shown below:

One non-limiting example of an activated 650 Compound 1/2 is thetetrafluorophenyl (TFP)-ester of 650 Compound 1/2, shown below:

One non-limiting example of an activated 650 Compound 1/2 is thesulfotetrafluorophenyl (STP)-ester of 650 Compound 1/2, shown below:

One non-limiting example of an activated 650 Compound 1/2 is thehydrazide of 650 Compound 1, shown below:

One non-limiting example of an activated 650 Compound 1/2 is themaleimide of 650 Compound 1, shown below:

In one embodiment, the compound is 650 Compound 2/2

650 Compound 2/2(2-((1E,3E,5E)-5-(1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methyl-5-sulfonatoindolin-2-ylidene)penta-1,3-dienyl)-1-(2-(2-methoxyethoxy)ethyl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonatetri sodium salt.) contains a (poly)ethylene glycol on the indole N ofthe left heterocycle. The methyl group on the ethylene glycol preventsthe terminal —OH from oxidation. Oxidation is known to occur, over time,on an unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 650 Compound 2/2 is activated as described above,one non-limiting example of which is the NHS-ester form of 650 Compound2/2, shown below.

In one embodiment, the compound is 650 Compound 3/2

650 Compound 3/2(2-((1E,3E,5E)-5-(1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methyl-5-sulfonatoindolin-2-ylidene)penta-1,3-dienyl)-1-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonatetri sodium salt) contains a (poly)ethylene glycol on the indole N of theleft heterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 650 Compound 3/2 is activated as described above,one non-limiting example of which is the NHS-ester form of 650 Compound3/2, shown below.

In one embodiment, the compound is 650 Compound 4/2

650 Compound 4/2(2-((1E,3E,5E)-5-(1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methyl-5-sulfonatoindolin-2-ylidene)penta-1,3-dienyl)-3-methyl-3-(3-sulfonatopropyl)-1-(2,5,8,11-tetraoxatridecan-13-yl)-3H-indolium-5-sulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 650 Compound 4/2 is activated as described above.

In one embodiment, the compound is 650 Compound 5/2

650 Compound 5/2(2-((1E,3E,5E)-5-(1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methyl-5-sulfonatoindolin-2-ylidene)penta-1,3-dienyl)-1-(2,5,8,11,14-pentaoxahexadecan-16-yl)-3-methyl-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 650 Compound 5/2 is activated as described above.

In one embodiment, the compound is 650 Compound 6/2

650 Compound 6/2(2-((1E,3E,5E)-5-(1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methyl-5-sulfonatoindolin-2-ylidene)penta-1,3-dienyl)-3-methyl-1-(2,5,8,11,14,17-hexaoxanonadecan-19-yl)-3-(3-sulfonatopropyl)-3H-indolium-5-sulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 650 Compound 6/2 is activated as described above.

In embodiments, the compound contains one or more substitutions of thepolymethine linker. In one embodiment, the compound has general formulaVa

general formula Vb

general formula Vc

or general formula Vd

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P-L-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a (poly)ethylene glycol group, where the(poly)ethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P-L-Z, and a caboxamidegroup -L-CONH—P-L-Z; each of R⁷ and R⁸ is the same or different and isindependently selected from either H, SO₃, a PEG group P-L-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and a(poly)ethylene glycol group where the (poly)ethylene glycol group is(CH₂CH₂O)_(s) where s is an integer from 3-6 inclusive, a sulfonamidegroup —SO₂NH—P-L-Z, or a caboxamide group —CONH—P-L-Z; where L isselected from the group consisting of a divalent linear (—(CH₂)_(o)—,o=0 to 15), crossed, or cyclic alkane group that can be substituted byat least one atom selected from the group consisting of oxygen,substituted nitrogen, and/or sulfur; where Z is selected from the groupconsisting of H, CH₃, alkyl, a heteroalkyl group, NH₂, —COO⁻, —COOH,—COSH, CO—NH—NH₂, —COF, —COCl, —COBr, —COI, —COO—Su(succinimidyl/sulfo-succinimidyl), —COO—STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′—CO—CH₂-1, —CONR′R″, —CONR′-biomolecule, —CONR′-L-COO⁻,—CONR′-L-COOH, —CONR′-L-COO—Su, —CONR′-L-COO—STP, —CONR′-L-COO-TFP,—CONR′-L-CONR″₂, —CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂,—CONR′-L-OH, —CONR′-L-O-phosphoramidite, —CONR′-L-CHO,—CONR′-L-maleimide, and —CONR′-L-NH—CO—CH₂—I; R′ and R″ is selected fromthe group consisting of H, aliphatic group, and heteroaliphatic group,and the biomolecule is a protein, antibody, nucleotide, oligonucleotide,biotin, or hapten; X is selected from the group consisting of —OH, —SH,—NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHS(hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O—STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH2-I, where R is —H or an aliphatic or heteroaliphaticgroup; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; p is an integer from 1 to 6 inclusive;each of R3 and R4 is the same or different and is independentlyhydrogen, an aliphatic group, a heteroaliphatic group, or a PEG groupP-L-Z where P is selected from an ethylene glycol group, a diethyleneglycol group, and a (poly)ethylene glycol group, where the(poly)ethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive; or R3 and R4 together form a cyclic structure where R3and R4 are joined using a divalent structural element selected from thegroup consisting of —(CH₂)_(q)—, —(CH₂)_(q)O(CH₂)_(q′)—,—(CH₂)_(q)S(CH₂)_(q′)—, —(CH₂)_(q)CH═CH—, —OCH═CH— where each of q andq′ is the same or different and is a integer from 2 to 6 inclusive; andY is selected from the group consisting of hydrogen, alkyl, sulfoalkyl,fluorine, chlorine, bromine, and a PEG group P-L-Z where P is selectedfrom an ethylene glycol group, a diethylene glycol group, and a(poly)ethylene glycol group, where the (poly)ethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive; with theproviso that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ containsa PEG group.

In one embodiment, the compound of general formula V wherein each of R3and R4 is the same or different and is independently hydrogen, analiphatic group, or a heteroaliphatic group, or R3 and R4 together forma cyclic structure where R3 and R4 are directly joined or joined using adivalent structural element selected from the group consisting of—(CH₂)_(q)— and CH═CH, where q is an integer from 1 to 2 inclusive, toresult in a 3-, 4-, or 5-membered ring.

In one embodiment, an isolated enantiomeric mixture selected fromdiastereomer Ia of general formula Va

diastereomer Ib of general formula Va

diastereomer Ic of general formula Vb

or diastereomer of general formula Vb

is provided, where each of R¹, R², R⁵, and R⁶ is the same or differentand is independently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P-L-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a (poly)ethylene glycol group, where the(poly)ethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P-L-Z, and a caboxamidegroup -L-CONH—P-L-Z; each of R⁷ and R⁸ is the same or different and isindependently selected from either H, SO₃, a PEG group P-L-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and a(poly)ethylene glycol group where the (poly)ethylene glycol group is(CH₂CH₂O), where s is an integer from 3-6 inclusive, a sulfonamide group—SO₂NH—P-L-Z, or a caboxamide group —CONH—P-L-Z; where L is selectedfrom the group consisting of a divalent linear (—(CH₂)_(o)—, o=0 to 15),crossed, or cyclic alkane group that can be substituted by at least oneatom selected from the group consisting of oxygen, substituted nitrogen,and/or sulfur; where Z is selected from the group consisting of H, CH₃,alkyl, a heteroalkyl group, NH₂, —COO⁻, —COOH, —COSH, CO—NH—NH₂, —COF,—COCl, —COBr, —COI, —COO—Su (succinimidyl/sulfo-succinimidyl), —COO—STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′—CO—CH₂—I, —CONR′R″, —CONR′-biomolecule, —CONR′-L-COO⁻,—CONR′-L-COOH, —CONR′-L-COO—Su, —CONR′-L-COO—STP, —CONR′-L-COO-TFP,—CONR′-L-CONR″₂, —CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂,—CONR′-L-OH, —CONR′-L-O-phosphoramidite, —CONR′-L-CHO,—CONR′-L-maleimide, and —CONR′-L-NH—CO—CH₂—I; R′ and R″ is selected fromthe group consisting of H, aliphatic group, and heteroaliphatic group,and the biomolecule is a protein, antibody, nucleotide, oligonucleotide,biotin, or hapten; X is selected from the group consisting of —OH, —SH,—NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHS(hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O—STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH2-I, where R is —H or an aliphatic or heteroaliphaticgroup; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive;each of R3 and R4 is the same or different and is independentlyhydrogen, an aliphatic group, a heteroaliphatic group, or a PEG groupP-L-Z where P is selected from an ethylene glycol group, a diethyleneglycol group, and a (poly)ethylene glycol group, where the(poly)ethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive; or R3 and R4 together form a cyclic structure where R3and R4 are joined using a divalent structural element selected from thegroup consisting of —(CH₂)_(q)—, —(CH₂)_(q)O(CH₂)_(q′)—,—(CH₂)_(q)S(CH₂)_(q′)—, —(CH₂)_(q)CH═CH—, —OCH═CH— where each of q andq′ is the same or different and is a integer from 2 to 6 inclusive; andY is selected from the group consisting of hydrogen, alkyl, sulfoalkyl,fluorine, chlorine, bromine, and a PEG group P-L-Z where P is selectedfrom an ethylene glycol group, a diethylene glycol group, and a(poly)ethylene glycol group, where the (poly)ethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive; with theproviso that at least one of R¹, R², R³, and R⁴ contains a PEG group.

In one embodiment, the compound of general formula V wherein each of R3and R4 is the same or different and is independently hydrogen, analiphatic group, or a heteroaliphatic group, or R3 and R4 together forma cyclic structure where R3 and R4 are directly joined or joined using adivalent structural element selected from the group consisting of—(CH₂)_(q)— and CH═CH, where q is an integer from 1 to 2 inclusive, toresult in a 3-, 4-, or 5-membered ring.

One non-limiting example is a substituted polymethine form of 650Compound 1/2, shown below:

One non-limiting example is a substituted polymethine form of 650Compound 2/2, shown below:

One non-limiting example is a substituted polymethine form of 650Compound 3/2, shown below:

One non-limiting example is a substituted polymethine form of 650Compound 4/2, shown below:

One non-limiting example is a substituted polymethine form of 650Compound 5/2, shown below:

One non-limiting example is a substituted polymethine form of 650Compound 6/2, shown below:

One non-limiting example is a substituted polymethine form of 650Compound 1/3 having an ethylene glycol, diethylene glycol, or(poly)ethylene glycol as described for general formula V, such as thecompound shown below:

One non-limiting example is a substituted polymethine form of 650Compound 1/2, shown below:

One non-limiting example is a substituted polymethine form of 650Compound 2/2, shown below:

One non-limiting example is a substituted polymethine form of 650Compound 3/2, shown below:

One non-limiting example is a substituted polymethine form of 650Compound 4/2, shown below:

One non-limiting example is a substituted polymethine form of 650Compound 5/2, shown below:

One non-limiting example is a substituted polymethine form of 650Compound 6/2, shown below:

One non-limiting example is a substituted polymethine form of 650Compound 1/3 having an ethylene glycol, diethylene glycol, or(poly)ethylene glycol as described for general formula V, such as thecompound shown below:

In embodiments, the degree of sulfonation is varied to, e.g., vary thecompound's degree of hydrophilicity or hydrophobicity. One non-limitingexample is a monosulfonate form of 650 Compound 1/2, shown below, but itis understood that the single sulfo group can be at any of the describedpositions:

One non-limiting example is a disulfonate form of 650 Compound 1/2,shown below, but it is understood that each of the two sulfo groups canbe at any of the described positions:

One non-limiting example is a trisulfonate form of 650 Compound 1/2,shown below, but it is understood that each of the three sulfo groupscan be at any of the described positions:

One non-limiting example is a tetrasulfonate form of 650 Compound 1/2,shown below, but it is understood that each of the four sulfo groups canbe at any of the described positions:

In various embodiments, an ethylene glycol group, diethylene glycolgroup, and/or a (poly)ethylene glycol group, which will collectively bereferred to as a PEG group, unless specifically defined, may be presentat position(s) in addition to such groups being present on the N atom(s)of the indole structure.

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R1 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R1 is adiethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R1 is a(poly)ethylene glycol (3) group terminating with a methyl

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R1 is a(poly)ethylene glycol (4) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R1 is a(poly)ethylene glycol (5) group terminating with a methyl

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R1 is a(poly)ethylene glycol (6) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R1 is asulfonamide group -L-SO₂NH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R1 is acarboxamide group -L-CONH—P—Z where Z is a methyl

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R2 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R2 is adiethylene glycol group terminating with a methyl group,

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R2 is a(poly)ethylene glycol (3) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R2 is a(poly)ethylene glycol (4) group terminating with a methyl

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R2 is a(poly)ethylene glycol (5) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R2 is a(poly)ethylene glycol (6) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R2 is asulfonamide group -L-SO₂NH—P—Z where Z is a methyl

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R2 is acarboxamide group -L-CONH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/3 according to general formula II where both R1 and R2are an ethylene glycol group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/3 according to general formula II where both R1 and R2are a diethylene glycol group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/3 according to general formula II where both R1 and R2are a (poly)ethylene glycol (3) group terminating with a methyl group,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/3 according to general formula II where both R1 and R2are a (poly)ethylene glycol (4) group terminating with a methyl group,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 4/4 according to general formula III where both R1 and R2are a (poly)ethylene glycol (4) group terminating with a methyl group,and R7 and R8 are sulfo, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 4/4 according to general formula III (V19-03005) whereboth R1 and R2 are a (poly)ethylene glycol (4) group terminating with amethyl group, and R7 and R8 are sulfo, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 4/4 according to general formula III where both R1 and R2are a (poly)ethylene glycol (4) group terminating with a methyl group,and R7 and R8 are H, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 4/4 according to general formula III where both R1 and R2are a (poly)ethylene glycol (4) group terminating with a methyl group,and R7 and R8 are H, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/3 according to general formula II where both R1 and R2are a (poly)ethylene glycol (5) group terminating with a methyl group,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/3 according to general formula II where both R1 and R2are a (poly)ethylene glycol (6) group terminating with a methyl group,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/3 according to general formula II where both R1 and R2are a sulfonamide group -L-SO₂NH—P—Z where Z is a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/3 according to general formula II where both R1 and R2are a carboxamide group -L-CONH—P—Z where Z is a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R8 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R8 issulfonamide —SO₂NH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R8 iscarboxamide —CONH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R7 is anethylene glycol group terminating with a methyl group,

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R7 is asulfonamide group —SO₂NH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/2 according to general formula II where R7 is acarboxamide group —CONH—P—Z where Z is a methyl group,

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/3 according to general formula II where both R7 and R8are an ethylene glycol group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/3 according to general formula II where both R7 and R8are a sulfonamide group —SO₂NH—P—Z where Z is a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 650 Compound 1/3 according to general formula II where both R7 and R8are a carboxamide group —CONH—P—Z where Z is a methyl group, shownbelow:

In one embodiment, the compound is 755 Compound 1/2

755 Compound 1/2(1-(5-carboxypentyl)-3-(2-methoxyethyl)-2-((1E,3E,5E,7E)-7-(1-(2-methoxyethyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)hepta-1,3,5-trienyl)-3-methyl-3H-indolium-5-sulfonatetri sodium salt) contains an ethylene glycol on the indole N of the leftheterocycle, i.e., a methylated ethylene glycol. The methyl group on theethylene glycol prevents the terminal —OH from oxidation. Oxidation isknown to occur, over time, on an unprotected PEG terminus (i.e., anunprotected terminus of an ethylene glycol group, diethylene glycolgroup, or (poly)ethylene glycol group). Adding a methyl ether providesthis protection, and prevents reaction with electrophilic reactivegroups.

In embodiments, e.g., for functional assays, the inventive compounds areactivated. Activation of the compound adds a chemical moiety such thatthe compound is in a form that can be conjugated to a biological moiety.Examples of chemical moieties for activation are described below withreference to activation of 755 Compound 1/2, but one skilled in the artappreciates that activation is not limited to these examples. Onenon-limiting example of an activated compound is the NHS-ester of 755Compound 1/2, shown below:

In one embodiment, the compound is an NHS-ester of 755 Compound 1/2where, according to general formula I, o is 1, shown below:

In one embodiment, the compound is an NHS-ester of 755 Compound 1/2where, according to general formula I, o is 5, shown below:

One non-limiting example of a NHS-ester of 755 Compound 1/3, accordingto general formula III, where m=1 and p=1, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 1/3, accordingto general formula III, where m=1 and p=2, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 1/3, accordingto general formula III, where m=1 and p=3, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 1/3, accordingto general formula III, where m=1 and p=4, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 1/3, accordingto general formula III, where m=1 and p=5, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 1/3, accordingto general formula III, where m=1 and p=6, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 2/3, accordingto general formula III, where m=2 and p=1, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 2/3, accordingto general formula III, where m=2 and p=2, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 2/3, accordingto general formula III, where m=2 and p=3, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 3/3, accordingto general formula III, where m=3 and p=1, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 3/3, accordingto general formula III, where m=3 and p=2, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 3/3, accordingto general formula III, where m=3 and p=3, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 4/3, accordingto general formula III, where m=4 and p=1, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 5/3, accordingto general formula III, where m=5 and p=1, is shown below:

One non-limiting example of a NHS-ester of 755 Compound 6/3, accordingto general formula III, where m=6 and p=1, is shown below:

One non-limiting example of an activated 755 Compound 1/2 is atetrafluorophenyl (TFP)-ester form of 755 Compound 1/2, shown below:

One non-limiting example of an activated 755 Compound 1/2 is asulfotetrafluorophenyl (STP)-ester form of 755 Compound 1/2, shownbelow:

One non-limiting example of an activated 755 Compound 1/2 is a hydrazideform of 755 Compound 1/2, shown below:

One non-limiting example of an activated 755 Compound 1/2 is a maleimideform of 755 Compound 1/2,

In one embodiment, the compound is 755 Compound 2/2

755 Compound 2/2(1-(5-carboxypentyl)-2-((1E,3E,5E,7E)-7-(1-(2-(2-methoxyethoxy)ethyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)hepta-1,3,5-trienyl)-3-(2-methoxyethyl)-3-methyl-3H-indolium-5-sulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 755 Compound 2/2 is activated as described above,one non-limiting example of which is the NHS-ester form of 755 Compound2/2, shown below.

In one embodiment, the compound is 755 Compound 3/2

755 Compound 3/2(1-(5-carboxypentyl)-2-((1E,3E,5E,7E)-7-(1-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)hepta-1,3,5-trienyl)-3-(2-methoxyethyl)-3-methyl-3H-indolium-5-sulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 755 Compound 3/2 is activated as described above,one non-limiting example of which is the NHS-ester form of 755 Compound3/2, shown below.

In one embodiment, the compound is 755 Compound 4/2

755 Compound 4/2(1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methyl-2-((1E,3E,5E,7E)-7-(3-methyl-5-sulfonato-3-(3-sulfonatopropyl)-1-(2,5,8,11-tetraoxatridecan-13-yl)indolin-2-ylidene)hepta-1,3,5-trienyl)-3H-indolium-5-sulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 755 Compound 4/2 is activated as described above.

In one embodiment, the compound is 755 Compound 5/2

755 Compound 5/2(2-((1E,3E,5E,7E)-7-(1-(2,5,8,11,14-pentaoxahexadecan-16-yl)-3-methyl-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)hepta-1,3,5-trienyl)-1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methyl-3H-indolium-5-sulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 755 Compound 5/2 is activated as described above.

In one embodiment, the compound is 755 Compound 6/2

755 Compound 6/2(1-(5-carboxypentyl)-3-(2-methoxyethyl)-3-methyl-2-((1E,3E,5E,7E)-7-(3-methyl-1-(2,5,8,11,14,17-hexaoxanonadecan-19-yl)-5-sulfonato-3-(3-sulfonatopropyl)indolin-2-ylidene)hepta-1,3,5-trienyl)-3H-indolium-5-sulfonate)contains a (poly)ethylene glycol on the indole N of the leftheterocycle. The methyl group on the ethylene glycol prevents theterminal —OH from oxidation. Oxidation is known to occur, over time, onan unprotected PEG terminus. Adding a methyl ether provides thisprotection, and prevents reaction with electrophilic reactive groups.For functional assays, 755 Compound 6/2 is activated as described above.

In embodiments, the compound contains one or more substitutions of thepolymethine linker. In one embodiment, the compound has general formulaVIa with “a” indicating an ethylene glycol, diethylene glycol, or(poly)ethylene glycol group on the left indole N, and the chain on theright indole N terminating in COX:

general formula VIb with “b” indicating an ethylene glycol, diethyleneglycol, or (poly)ethylene glycol group on the left indole N, and thechain on the right indole N terminating in COH:

general formula VIc with “c” indicating an ethylene glycol, diethyleneglycol, or (poly)ethylene glycol group on the left and right indole N,and the chain on the right indole N terminating in COX:

or general formula VId with “d” indicating an ethylene glycol,diethylene glycol, or (poly)ethylene glycol group on the left and rightindole N, and the chain on the right indole N terminating in COH:

where each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of an aliphatic,heteroaliphatic, sulfoalkyl group, heteroaliphatic with terminal SO₃, aPEG group P-L-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a (poly)ethylene glycol group, where the(poly)ethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P-L-Z, and a caboxamidegroup -L-CONH—P-L-Z; each of R⁷ and R⁸ is the same or different and isindependently selected from either H, SO₃, a PEG group P-L-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and a(poly)ethylene glycol group, where the (poly)ethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, a sulfonamidegroup —SO₂NH—P-L-Z, or a caboxamide group —CONH—P-L-Z; where L isselected from the group consisting of a divalent linear (—(CH₂)_(o)—,o=0 to 15), crossed, or cyclic alkane group that can be substituted byat least one atom selected from the group consisting of oxygen,substituted nitrogen, and/or sulfur; where Z is selected from the groupconsisting of H, CH₃, alkyl, a heteroalkyl group, NH₂, —COO⁻, —COOH,—COSH, CO—NH—NH₂, —COF, —COCl, —COBr, —COI, —COO—Su(succinimidyl/sulfo-succinimidyl), —COO—STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′—CO—CH₂—I, —CONR′R″, —CONR′-biomolecule, —CONR′-L-COO⁻,—CONR′-L-COOH, —CONR′-L-COO—Su, —CONR′-L-COO—STP, —CONR′-L-COO-TFP,—CONR′-L-CONR″₂, —CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂,—CONR′-L-OH, —CONR′-L-O-phosphoramidite, —CONR′-L-CHO,—CONR′-L-maleimide, and —CONR′-L-NH—CO—CH₂—I; R′ and R″ is selected fromthe group consisting of H, aliphatic group, and heteroaliphatic group,and the biomolecule is a protein, antibody, nucleotide, oligonucleotide,biotin, or hapten; X is selected from the group consisting of —OH, —SH,—NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHS(hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O—STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH2-I, where R is —H or an aliphatic or heteroaliphaticgroup; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; p is an integer from 1 to 6 inclusive;each of R3 and R4 is the same or different and is independentlyhydrogen, an aliphatic group, a heteroaliphatic group, or a PEG groupP-L-Z where P is selected from an ethylene glycol group, a diethyleneglycol group, and a (poly)ethylene glycol group, where the(poly)ethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive; or R3 and R4 together form a cyclic structure where R3and R4 are joined using a divalent structural element selected from thegroup consisting of —(CH₂)_(q)—, —(CH₂)_(q)O(CH₂)_(q′)—,—(CH₂)_(q)S(CH₂)_(q′)—, —(CH₂)_(q)CH═CH—, —OCH═CH— where each of q andq′ is the same or different and is a integer from 2 to 6 inclusive; andY is selected from the group consisting of hydrogen, alkyl, sulfoalkyl,fluorine, chlorine, bromine, a PEG group P-L-Z where P is selected froman ethylene glycol group, a diethylene glycol group, and a(poly)ethylene glycol group, where the (poly)ethylene glycol group is(CH₂CH₂O)_(s), where s is an integer from 3-6 inclusive, and anoxygen-containing group OR^(PM) where R^(PM) is selected from the groupconsisting of hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cyclic alkyl,substituted or unsubstituted heterocyclic alkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl, wherethe group can be substituted with at least one of hydroxyl, sulfo,carboxy, and/or amino; with the proviso that at least one of R¹, R², R³,R⁴, R⁵, R⁶, R⁷, and R⁸ contains a PEG group.

In one embodiment, the compound of general formula VI where each of R3and R4 is the same or different and is independently hydrogen, analiphatic group, or a heteroaliphatic group, or R3 and R4 together forma cyclic structure where R3 and R4 are directly joined or joined using adivalent structural element selected from the group consisting of—(CH₂)_(q)— and CH═CH, where q is an integer from 1 to 2 inclusive, toresult in a 3-, 4-, or 5-membered ring.

In one embodiment, the compound of general formula VI wherein R3 and R4together form a cyclic structure where R3 and R4 are joined using adivalent structural element of —(CH₂)_(q)—, where q is 3, to result in a6-membered ring, and Y is OR^(PM) where R^(PM) is a substituted6-membered aryl group, where the substituted group is sulfo.

One non-limiting example is a substituted polymethine form of 755Compound 1/2, shown below:

One non-limiting example is a substituted polymethine form of 755Compound 2/2, shown below:

One non-limiting example is a substituted polymethine form of 755Compound 3/2, shown below:

One non-limiting example is a substituted polymethine form of 755Compound 4/2, shown below:

One non-limiting example is a substituted polymethine form of 755Compound 5/2, shown below:

One non-limiting example is a substituted polymethine form of 755Compound 6/2, shown below:

One non-limiting example is a substituted polymethine form of 755Compound 1/3 having an ethylene glycol, diethylene glycol, or(poly)ethylene glycol as described for general formula VI, such as thecompound shown below:

One non-limiting example is a substituted polymethine form of 755Compound 4/4 having an ethylene glycol, diethylene glycol, or(poly)ethylene glycol as described for general formula VI, such as thecompound shown below:

In embodiments, the degree of sulfonation is varied to, e.g., vary thecompound's degree of hydroplilicity or hydrophobicity. One non-limitingexample is a monosulfonate form of 755 Compound 1/2, shown below, but itis understood that the single sulfo group can be at any of the describedpositions:

One non-limiting example is a disulfonate form of 755 Compound 1/2,shown below, but it is understood that the each of the two sulfo groupscan be at any of the described positions:

One non-limiting example is a trisulfonate form of 755 Compound 1/2,shown below, but it is understood that the each of the three sulfogroups can be at any of the described positions:

One non-limiting example is a tetrasulfonate form of 755 Compound 1,shown below, but it is understood that the each of the four sulfo groupscan be at any of the described positions:

In embodiments, an ethylene glycol group, diethylene glycol group,and/or a (poly)ethylene glycol group, which will collectively bereferred to as a PEG group unless specifically defined, may be presentat position(s) in addition to such groups being present on the N atom(s)of the indole structure. One non-limiting example of an additionallyPEG-substituted compound is a 755 Compound 1/2 according to generalformula II where R1 is an ethylene glycol group terminating with amethyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R1 is adiethylene glycol group terminating with a methyl group,

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R1 is a(poly)ethylene glycol (3) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R1 is a(poly)ethylene glycol (4) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R1 is a(poly)ethylene glycol (5) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R1 is a(poly)ethylene glycol (6) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R1 is asulfonamide group -L-SO₂NH—P—Z where Z is a methyl

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R1 is acarboxamide group -L-CONH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R2 is anethylene glycol group terminating with a methyl group,

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R2 is adiethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R2 is a(poly)ethylene glycol (3) group terminating with a methyl

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R2 is a(poly)ethylene glycol (4) group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R2 is a(poly)ethylene glycol (5) group terminating with a methyl

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R2 is a(poly)ethylene glycol (6) group terminating with a methyl

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R2 is asulfonamide group -L-SO₂NH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R2 is acarboxamide group -L-CONH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/3 according to general formula II where both R1 and R2are an ethylene glycol group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/3 according to general formula II where both R1 and R2are a diethylene glycol group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/3 according to general formula II where both R1 and R2are a (poly)ethylene glycol (3) group terminating with a methyl group,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/3 according to general formula II where both R1 and R2are a (poly)ethylene glycol (4) group terminating with a methyl group,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 4/4 according to general formula II where both R1 and R2are a (poly)ethylene glycol (4) group terminating with a methyl group,and R7 and R8 are sulfo, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 4/4 according to general formula II where both R1 and R2are a (poly)ethylene glycol (4) group terminating with a methyl group,and R7 and R8 are H, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/3 according to general formula II where both R1 and R2are a (poly)ethylene glycol (5) group terminating with a methyl group,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/3 according to general formula II where both R1 and R2are a (poly)ethylene glycol (6) group terminating with a methyl group,shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/3 according to general formula II where both R1 and R2are a sulfonamide group -L-SO₂NH—P—Z where Z is a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/3 according to general formula II where both R1 and R2are a carboxamide group -L-CONH—P—Z where Z is a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R8 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R8 issulfonamide —SO₂NH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R8 iscarboxamide —CONH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R7 is anethylene glycol group terminating with a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R7 is asulfonamide group —SO₂NH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/2 according to general formula II where R7 is acarboxamide group —CONH—P—Z where Z is a methyl group, shown below:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/3 according to general formula II where both R7 and R8are an ethylene glycol group terminating with a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/3 according to general formula II where both R7 and R8are a sulfonamide group —SO₂NH—P—Z where Z is a methyl group, shownbelow:

One non-limiting example of an additionally PEG-substituted compound isa 755 Compound 1/3 according to general formula II where both R7 and R8are a carboxamide group —CONH—P—Z where Z is a methyl group, shownbelow:

The disclosed compounds are used and are useful as chromophores and/orfluorophores. For example, they can be used for optical labelling and,therefore, for the qualitative and/or quantitative detection ofproteins, nucleic acids, oligomers, DNA, RNA, biological cells, lipids,mono-, oligo- and polysaccharides, ligands, receptors, polymers, drugs,polymeric beads, etc.

The inventive compounds, containing the disclosed functionality orfunctionalities, may be synthesized using methods known in the art,e.g., as described as follows with all references expressly incorporatedby reference herein in their entirety. The hydrophilicity orhydrophobicity of the inventive compounds is modified by the number andlocation of hydrophilic groups, such as sulfo, carboxy, hydroxy, etc.,groups. In embodiments, the number and location of hydrophilic groups issymmetrical, such that the number and location of hydrophilic group(s)on one of the indoles of the inventive cyanine compound is also found onthe other indole. In various embodiments, at least one hydrophilic groupis found on each of the indoles of the inventive cyanine compound.Similarly, solubility, lack of aggregation, reactivity, lack ofcross-reactivity, etc., are effected by the number and location of thedisclosed functionality or functionalities on the compound.

In one embodiment, short PEG groups are added on opposite sides andopposite ends of indole cyanine compounds to effectively surround thehydrophobic core structure of the molecule. In another embodiment,sulfonate groups are added to the outer phenyl rings of indole cyaninedyes along with the symmetrical placement of short PEG chains onopposite sides and opposite ends.

Adding PEG 1-6, if at the appropriate positions to strategicallysurround the core dye structure, have significant beneficial effects onthe hydrophilicity and performance of these dyes in biologicalapplications. Previous attempts to make dyes more hydrophilic and less“sticky” toward biomolecules included the addition of multiplesulfonates or much longer PEG chains to some locations on dye molecules.However, the addition of too many sulfonates, while having the effect ofincreasing the relative water solubility of dyes, can create undesirablenonspecific binding character due to negative charge interactions withpositively charged biomolecules, particularly proteins. In addition,previous attempts to make dyes more water soluble by adding longer PEGchains to one or two sites on a dye has the detrimental effect ofdramatically increasing the molecular weight of the dye, possiblypreventing efficient access of dye-labeled antibodies and otherdye-labeled targeting molecules to bind with inner cellular targets,while also not fully surrounding and masking the hydrophobic dye corestructure. The inventors have discovered that by using short PEG chainmodifications at critical sites on a dye structure that the totalmolecular size of labeled molecules can be limited, while nonspecificityis dramatically reduced by masking the hydrophobic dye core.

In one embodiment, PEG 1-6 group(s) are added on opposite sides andopposite ends of indole cyanine compounds to effectively surround thehydrophobic core structure of the molecule. In another embodiment,sulfonate groups are added to the outer phenyl rings of indole cyaninedyes along with the symmetrical placement of short PEG chains onopposite sides and opposite ends.

The core indocyanine structure without additional functionalilities,along with its synthesis, was described by König in U.S. Pat. No.1,524,791 and BP 434875, and included 3-, 5-, and 7-membered polymethinechains.

Synthesis of numerous modifications of the core indocyanine structurehave been described. Such modifications provided variousfunctionalilities, e.g., synthesis of N-isothiocyanato-alkyl- andaromatic-carboxyalkyl-functionalized indocyanines were described in U.S.Pat. Nos. 5,627,027; 6,048,982; 4,981,977; U.S. Publication No.2006/0199949; Southwick, Anal. Chem. 67 (1995) 1742-48).

Synthesis of indocyanines with one or two N-carboxyalkyl functionalitieswere described in U.S. Pat. Nos. 5,268,486; 5,486,616; 5,569,587;5,569,766; JP 03217837.

Synthesis of indocyanines containing C-carboxyalkyl groups weredescribed in JP 05-313304; U.S. Publication Nos. 2006/0099638,2006/0004188; 2002/0077487; 2002/0064794; U.S. Pat. Nos. 6,977,305 and6,974,873.

Synthesis of indocyanines with N- and C-sulfoalkyl groups were describedin JP 05-313304; WO 2005/044923; U.S. Publication No. 2007/0203343.

Synthesis of indocyanines with mixed C-carboxyalkyl and C-sulfoalkylwere described in EP 1792949 and U.S. Pat. No. 7,745,640.

Synthesis of indocyanaines having a PEG-containing, N-carboxyalkylspacer were described in U.S. Pat. No. 6,939,532.

Functionalization of the N-carboxyalkyl with an amino-functionalizedPEG-alkyl chain, and N- and C-substituted PEG-alkyl chains, weredescribed in U.S. Publication No. 2009/0305410.

Synthesis of various polymethine bridge substitutions, and otherfunctionalizations of indocyanines, were described in Strekowski,Heterocyclic Polymethine Dyes: Synthesis, Properties and Applications,(2008) Springer-Verlag, Berlin Heidelberg; Gragg, “Synthesis ofNear-Infrared Heptamethine Cyanine Dyes” (2010). Chemistry Theses. Paper28. http://digitalarchive.gsu.edu/chemistry_theses/28; Patonay et al.(2004) Noncovalent Labeling of Biomolecules with Red and Near-InfraredDyes, Molecules 9 (2004) 40-49; and U.S. Pat. No. 7,172,907.

In one embodiment, the compound is synthesized by a condensationreaction, known to one skilled in the art, of the two differentlysubstituted indole heterocycles separated by a (poly)methine linker orbridge, e.g., C1, C3, or C5. Other synthesis methods are possible. Asonly one example, one of the indole heterocycles is first reacted withthe C1, C3, or C5 linker. The 1:1 condensation product is isolated, andthen condensed with the second indole heterocycle to result in thecyanine compound. The sequence of reacting the indole heterocycles isirrelevant. Thus, a plurality of differently functionalized, stronglyhydrophilic, diastereomeric compounds that differ in total charge andspecificity/reactivity of the active groups used for theirimmobilization, were prepared.

Conjugates of the compounds were prepared by covalently coupling thecompounds to a biomolecule using the functional substituent on theN-position of the indole ring. This functional substituent was activatedby routine protein chemistry reaction methods known to one skilled inthe art. The activated compound may be converted to, e.g, and withoutlimitation, aN-hydroxysuccinimide (NHS)-ester, an acid fluoride, atetrafluorophenyl (TFP)- or sulfotetrafluorophenyl (STP)-ester, aniodoacetyl group, a maleimide, a hydrazide, a sulfonyl chloride, or aphenylazide. Methods for preparing such compounds are known to oneskilled in the art. In one embodiment, the activated substituent wasthen reacted with an amino group on the biomolecule under conditions toconjugate the desired biomolecule.

In one embodiment, a non-activated carboxyl group on the N-position ofthe indole in the compound was coupled to an amine using a carbodimide.

In one embodiment, a N-hydroxysuccinimidyl ester (X=—NHS) of a compoundwas formed as follows: 20 μmol dye with X═OH (carboxyalkyl group), 8 mg(40 μmol) dicyclohexylcarbodiimide, and 5 mg (40 μmol)N-hydroxysuccinimide were dissolved in 2 ml DMF and 100 μl water. Six μl(40 μmol) triethylamine was added. The reaction mixture was stirred atroom temperature (about 20° C. to about 22° C.) for 24 hours and thenfiltered. The solvent was removed and the residue was washed withdiethylether. The reaction proceeded quantitatively.

In one embodiment, a maleimide (X=—NH—CH₂CH₂-maleimide) of a compound isformed as follows: 20 μmol dye with X=—NHS (N-hydroxysuccinimid-ester)was dissolved in 2 ml DMF and 100 μl water and mixed with 7.6 mg (30μmol) 2-maleimidoethylamine-trifluoracetate and 5 μl (30 μmol)N-ethyldiisopropyl-amine. The reaction mixture is stirred for 3 h atroom temperature (about 20° C. to about 22° C.). The solvent wasevaporated under reduced pressure. The residue is washed withdiethylether and acetone and dried in vacuum. The reaction proceedsquantitatively.

In one embodiment, a iodoacetamide (X=—NH—CH₂CH₂—NH—CO—CH₂—I) of acompound is formed as follows: 20 μmol dye with X=—NHS(N-hydroxysuccinimid-ester) was dissolved in 2 ml DMF and 100 μl water,followed by addition of 40 mg (300 μmol) ethylendiamindihydrochlorideand 26 μl (150 μmol) N-ethyldiisopropyl-amine. The reaction mixture isstirred for 3 h at room temperature (about 20° C. to about 22° C.). Thesolvent is then evaporated under reduced pressure, the residue wasdissolved in methanol, and the ethylendiamindihydrochlorid was removedby filtration. The methanol is evaporated under reduced pressure. Theresidue is dissolved in 2 ml dry DMF, followed by addition of 7 mg (25μmol) N-succinimidyl iodoacetate and 4 μl (25 μmol)N-ethyldiisopropylamine. The reaction mixture is stirred for 3 h at roomtemperature. The solvent was evaporated under reduced pressure and theresidue was purified by reverse phase HPLC.

In one embodiment, a hydroxyl group, such as a terminal hydroxyl group,can be subsequently activated to a reactive derivative able to linkwith, for example, proteins and other molecules. Examples of activatinggroups include tosyl chloride (TsCl), tresyl chloride (TrCl),disuccinimidyl carbonate (DSC), divinyl sulfone, bis-epoxy compounds,carbonyl diimidazole (CDI), 2-fluoro-1-methylpyridinium (FMP), andtrichloro-s-triazine (TsT). In one embodiment, the hydroxyl group isactivated to a succinimidyl carbonate, which is reactive with amines.

Coupling between the compound and the biomolecule may be performed asfollows. The compound was reacted with the biomolecule in an organic oraqueous solution at pH between pH 5-pH 12 inclusive. The compound neednot be dissolved in an organic solvent, such as dimethyl formamide (DMF)or dimethyl sulfoxide (DMSO) prior to adding the biomolecule. In oneembodiment, coupling reaction may be performed in a 100% aqueoussolution. In one embodiment, the coupling reaction occurs at roomtemperature (about 20° C. to about 22° C.).

To form a composition (dye), at least one biocompatible excipient wasadded to the compound(s), as known to one of ordinary skill in the art.Excipients include, but are not limited to, buffers, solubilityenhancing agents, stabilizing agents, etc.

In one embodiment, a kit for performing an assay method comprises adisclosed compound, and instructions for performing the method using thecompound.

The disclosed activated compounds (i.e., the compound modified with areactive group) are useful to label macromolecules (e.g., antibodies,streptavidin, etc) using methods known to one skilled in the art, e.g.,Hermanson, Bioconjugate Techniques, 2nd Ed., London, Elsevier Inc. 2008.The reaction was carried out for 1-2 h at room temperature (about 20° C.to about 22° C.), and then desalted by dialyzing against several changesof phosphate buffered saline (pH 7.2) or purified by gel filtration toremove the unreacted fluorescent dye. The resulting compound-biomoleculeconjugate was used to detect, e.g., specific proteins in immunoassays,sugars in glycoproteins with lectins, protein-protein interactions,oligonucleotides in nucleic acid, hybridization, and in electrophoreticmobility shift assays (EMSA).

The resulting compound-biomolecule conjugates exhibited fluorescentproperties. In this embodiment, they were used in optical methodsincluding fluorescence opticalqualitative and quantitative determinationmethods. Examples of such methods include, but are not limited to,microscopy, immunoassays, hybridization methods, chromatographic andelectrophoretic methods, fluorescence resonance energy transfer (FRET)systems, bioluminescence reasonance energy transfer (BRET), highthroughput screenings, analysis of receptor-ligand interactions on amicroarray, etc.

Compounds in any embodiment were used as dyes for optical labelling oforganic or inorganic biomolecules, referred to as recognition units.Recognition units are molecules having specificity and/or affinity for aspecific group of molecules. Examples include, but are not limited to,antibodies that have affinity for antigens, enzymes that bind and/orreact with a specific bond or bonds within a sequence of amino acids ina peptide or react with a substrate, cofactors such as metals thatenhance or inhibit specific interactions, lectins that bind specificsugars or sugar sequences (e.g., oligosaccharides, polysaccharides,dextrans, etc.), biotin binding proteins such as avidin and streptavidinthat bind biotin and biotinylated molecules, antibody binding proteinssuch as Protein A, Protein G, Protein A/G and Protein L, sequences ofamino acids or metals that have affinity for each other (e.g., histidinesequences that bind nickel or copper, phosphate containing proteins thatbind gallium, aluminium, etc.), specific sequences of nucleic acids suchas DNA and/or RNA oligonucleotides that have affinity for proteins,specific sequences of amino acids that have affinity for DNA and/or RNA,haptens, carotenoids, hormones (e.g., neurohormones), neurotransmitters,growth factors, toxins, biological cells, lipids, receptor binding drugsor organic or inorganic polymeric carrier materials, fluorescentproteins such as phycobilliproteins (e.g., phycoethrin,allophycocyanin), etc. Ionic interactions between recognition units andthe disclosed compounds results in labeling of the recognition units.The recognition unit and compound can be covalently bound. The result isa conjugate for qualitative or quantitative determination of variousbiomaterials or other organic or inorganic materials using opticalmethods.

The inventive compounds and/or conjugates are used in optical, includingfluorescence optical, qualitative and/or quantitative determinationmethods to diagnose properties of cells (molecular imaging), inbiosensors (point of care measurements), for investigation of thegenome, and in miniaturizing technologies. Microscopy, cytometry, cellsorting, fluorescence correlation spectroscopy (FCS), ultra highthroughput screening (uHTS), multicolor fluorescence in situhybridisation (mc-FISH), FRET-systems, BRET-systems, and microarrays(DNA- and protein-chips) are exemplary application fields. As known toone skilled in the art, a microarray is a grid-like arrangement wheremore than two different molecules are immobilized in a known predefinedregion on at least one surface, and is useful to evaluate receptorligand interactions. As known to one skilled in the art, a receptor is anaturally occurring or synthetic molecule that exhibits an affinity to agiven ligand. Receptors can be used in a pure form or bound to anotherspecie. Receptors can be coupled covalently or noncovalently to abinding partner either directly or indirectly (e.g., through a couplingmediator). Receptor examples include, but are not limited to, agonistsand antagonists for cell membrane receptors, toxins and other poisons,viral epitopes, hormones (e.g., opiates, steroids), hormone receptors,peptides, enzymes, enzyme substrates, drugs acting as cofactors,lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides,cells, cell fragments, tissue fragments, proteins, antibodies, etc. Asknown to one skilled in the art, a ligand is a molecule that isrecognized by a certain receptor. Ligand examples include, but are notlimited to, agonists and antagonists for cell membrane receptors, toxinsand other poisons, viral epitopes, hormones (e.g., opiates, steroids),hormone receptors, peptides, enzymes, enzyme substrates, drugs acting ascofactors, lectins, sugars, oligonucleotides, nucleic acids,oligosaccharides, proteins, antibodies, etc.

The following non-limiting examples further describe the compounds,methods, compositions, uses, and embodiments.

EXAMPLE 1 Synthesis of 4-methyl-5-oxohexane sulfonic acid used tosynthesize EXAMPLE 2 compound2,3-dimethyl-3-(3-sulfopropyl)-3H-indole-5-sulfonic acid di-potassiumsalt and EXAMPLE 8 compound1,2-dimethyl-1-(3-sulfopropyl)-1H-benzo[e]indole-6,8-disulfonic acidtripotassium salt

Sodium hydride (2.1 g, 80 wt %=69 mmol) was slurried in 10 ml of dryTHF. The suspension was cooled to 0° C. and a solution ofethyl-2-methylacetoacetate (10 g, 69 mmol) in 10 ml of dry THF was addeddropwise. The solution was stirred at room temperature for 1 h. Asolution of 1,3-propanesultone (8.42 g, 69 mmol) in 10 ml of dry THF wasadded dropwise. Once the addition was complete, the solution was stirredfor 2 h at 40° C. The solution was evaporated to dryness. The residuewas dissolved in 100 ml water. The aqueous solution was extracted twicewith ethylacetate, then 100 ml concentrated HCl was added and thesolution was refluxed for 2 h. The solvent was evaporated in vacuum. Theresidue was purified by column chromatography (silica,methanol/dichloromethane) to give 4-methyl-5-oxohexane sulfonic acid.Yield 10 g; MS (ESI−): 193.2 [M]⁻

EXAMPLE 2 Synthesis of2,3-dimethyl-3-(3-sulfopropyl)-3H-indole-5-sulfonic acid di-potassiumsalt used to synthesize EXAMPLE 3 compound1-(2-methoxy-ethyl)-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumand EXAMPLE 4 compound1-[2-(2-methoxy-ethoxy)-ethyl]-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumand EXAMPLE 5 compound1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumand EXAMPLE 6 compound1-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indolium

Ten g (51 mmol) 4-hydrazino-benzene sulfonic acid and 9.85 g (51 mmol)4-methyl-5-oxohexane sulfonic acid were dissolved in 50 ml acetic acid.The solution was heated at 140° C. for 4 h. The solvent was evaporatedin vacuum. The oily residue was dissolved in 20 ml methanol, then 50 mlof a saturated solution of KOH in 2-propanol was added to yield a yellowprecipitate. The solid was filtered off and dried in vacuum. Yield 11 g,MS (ESI−): 172.5 [M]²⁻

EXAMPLE 3 Synthesis of1-(2-methoxy-ethyl)-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumused to synthesize 550, 650, 755 Compound 1

A mixture of 5 g (12.4 mmol)2,3-dimethyl-3-(3-sulfopropyl)-3H-indole-5-sulfonic acid dipotassiumsalt and 5.89 g (25.6 mmol) 2-methoxyethyl-p-toluene sulfonate washeated under argon for 24 h. The residue was purified by columnchromatography (reversed phase silica, methanol/water, TFA). Yield 2.3g, MS (ESI−): 404.1 [M-H]⁻

EXAMPLE 4 Synthesis of1-[2-(2-methoxy-ethoxy)-ethyl]-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumused to synthesize 550, 650, 755 Compound 2

A mixture of 5 g (12.4 mmol)2,3-dimethyl-3-(3-sulfopropyl)-3H-indole-5-sulfonic acid dipotassiumsalt and 7.1 g (25.6 mmol) [2-(2-methoxyethoxy)ethoxy]-p-toluenesulfonate was heated under argon for 24 h. The residue was purified bycolumn chromatography (reversed phase silica, methanol/water, TFA).Yield 2.0 g. MS (ESI−): 448.2 [M-H]⁻

EXAMPLE 5 Synthesis of1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumused to synthesize 550, 650, 755 Compound 3

A mixture of 5 g (12.8 mmol)2,3-dimethyl-3-(3-sulfopropyl)-3H-indole-5-sulfonic acid dipotassiumsalt and 8.14 g (25.6 mmol)[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]p-toluene sulfonate was heatedunder argon for 24 h. The residue was purified by column chromatography(reversed phase silica, methanol/water, TFA). Yield 1.9 g, MS (ESI−):492.1 [M-H]⁻

EXAMPLE 6 Synthesis of1-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumused to synthesize EXAMPLE 7 compound1-(5-carboxypentyl)-3-methyl-2-((E)-2-phenylamino-vinyl)-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumand EXAMPLE 8 compound1-(5-carboxypentyl)-3-methyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand EXAMPLE 9 compound1-(5-carboxypentyl)-3-methyl-2-((1E,3E,5E)-6-phenylamino-hexa-1,3,5-trienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indolium

Both 5 g (15.7 mmol) 6-hydrazino-naphthalene-1,3-disulfonic acid and4.93 g (25 mmol) 4-methyl-5-oxohexane sulfonic acid were dissolved in 50ml acetic acid. The solution was heated at 140° C. for 4 h. The solventwas evaporated in a vacuum. The oily residue was dissolved in 20 mlmethanol, then 50 ml of a saturated solution of KOH in 2-propanol wasadded to yield a yellow precipitate. The solid was filtered off anddried in vacuum. Yield 4.1 g, MS (ESI−): 158.2 [M]³⁻

EXAMPLE 7 Synthesis of1-(5-carboxypentyl)-3-methyl-2-((E)-2-phenylamino-vinyl)-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumused to synthesize 550 Compounds

A combination of 0.92 g (2 mmol)1-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 0.43 g (2.2 mmol) N,N′-diphenylformamidine was dissolved in 20 mlmethanol and stirred for 4 h under reflux. The solvent was removed undervacuum. The residue was washed carefully with ethyl acetate. A darkyellow solid was obtained which was processed without furtherpurification. MS (ESI−): 563.1 [M-H]⁻

EXAMPLE 8 Synthesis of1-(5-carboxypentyl)-3-methyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumused to synthesize 650 Compounds

A combination of 0.92 g (2 mmol)1-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 0.57 g (2.2 mmol) malonaldehyde-bisphenylimine-hydrochloride weredissolved in 10 ml acetic acid and 10 ml acetic anhydride and stirredfor 4 h at 120° C. The solvent was removed under vacuum. The residue waswashed carefully with ethyl acetate. A dark brown solid was obtainedwhich was processed without further purification. MS (ESI−): 589.2[M-H]⁻

EXAMPLE 9 Synthesis of1-(5-carboxypentyl)-3-methyl-2-((1E,3E,5E)-6-phenylamino-hexa-1,3,5-trienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumused to synthesize 755 Compounds

A combination of 0.92 g (2 mmol)1-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 0.63 g (2.2 mmol) glutacondianil-hydrochloride were dissolved in 10ml acetic acid and 10 ml acetic anhydride and stirred for 4 h at 120° C.The solvent was removed under vacuum. The residue was washed carefullywith ethyl acetate. A dark solid was obtained which was processedwithout further purification. MS (ESI−): 615.2 [M-H]⁻

EXAMPLE 10 Synthesis of 550 Compound 12-{(E)-3-[1-(5-carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-propenyl}-1-(2-methoxy-ethyl)-3-methyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumtri sodium salt

Five hundred sixty-four mg (1 mmol)1-(5-carboxypentyl)-3-methyl-2-((E)-2-phenylamino-vinyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 404 mg (1 mmol)1-(2-methoxy-ethyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumwere dissolved in 20 ml of acetic acid/acetic anhydride (1/1), followedby 200 mg sodium acetate. The solution was stirred under reflux for 15min. After cooling to room temperature, 20 ml diethylether was added.The resulting precipitate (mixture of the diastereomers 550 Compound 1(isomer 1) and 550 Compound 1 (isomer 2)) was extracted by suction,washed with ether, and dried.

The residue was purified by column chromatography (RP-18,acetonitrile/water and concentrated HCl) to separate the diastereomersfrom each other. The diastereomer that first eluted from the column wastermed diastereomer 1 (550 Compound 1 (isomer 1)). The diastereomer thateluted second from the column was termed diastereomer 2 (550 Compound 1(isomer 2)). The diastereomers were separated, followed byneutralization and evaporation. Purification of the singlediastereomeric compound was completed on a RP-18 column,acetonitrile/water. The corresponding fractions were pooled and thesolvent was removed by distillation. The two products (diastereomers 550Compound 1 (isomer 1) and 550 Compound 1 (isomer 2)) were dried in highvacuum.

550 Compound 1 (Isomer 1)

yield: 12%

UV-vis (PBS): λmax=557 nm, λem=572 nm

MS (ESI−) [M/z]: 291.2 [M]³⁻; 448.3 [M+Na]²⁻

550 Compound 1 (Isomer 2):

yield: 23%

UV-vis (PBS): λmax=557 nm, λem=572 nm

MS (ESI−) [M/z]: 291.1 [M]³⁻; 448.2 [M+Na]²⁻

EXAMPLE 11 Synthesis of 550 Compound 22-{(E)-3-[1-(5-carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-propenyl}-1-[2-(2-methoxy-ethoxy)-ethyl]-3-methyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumtri sodium salt

Both 1 mmol1-(5-carboxypentyl)-3-methyl-2-((E)-2-phenylamino-vinyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 1 mmol1-[2-(2-methoxy-ethoxy)-ethyl]-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumwere dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed bythe addition of 200 mg sodium acetate. The solution was stirred underreflux for 15 min. After cooling to room temperature, 20 ml diethyletherwas added. The resulting precipitate (mixture of the diastereomers 550-1compound 2 and 550-2 compound 2) was extracted by suction, washed withether and dried.

The residue was purified by column chromatography (RP-18,acetonitrile/water and concentrated HCl), thereby separating thediastereomers from each other, as described in Example 10.

EXAMPLE 12 Synthesis of 550 Compound 32-{(E)-3-[1-(5-carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-propenyl}-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-3-methyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumtri sodium salt

One mmol1-(5-carboxypentyl)-3-methyl-2-((E)-2-phenylamino-vinyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 1 mmol1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumwere dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed bythe addition of 200 mg sodium acetate. The solution was stirred underreflux for 15 min. After cooling to room temperature, 20 ml diethyletherwas added. The resulting precipitate (mixture of the diastereomers 550-1compound 2 and 550-2 compound 2) was extracted by suction, washed withether and dried.

The residue was purified by column chromatography (RP-18,acetonitrile/water and concentrated HCl), thereby separating thediastereomers from each other, as described in Example 10.

EXAMPLE 13 650 Compound 1 Synthesis of2-{(1E,3E)-5-[1-(5-carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-penta-1,3-dienyl}-1-(2-methoxy-ethyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumtri sodium salt

Both 90 mg (1 mmol)1-(5-carboxypentyl)-3-methyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 404 mg (1 mmol)1-(2-methoxy-ethyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumwere dissolved in 20 ml of acetic acid/acetic anhydride (1/1) followedby the addition of 200 mg of sodium acetate. The solution was stirredunder reflux for 15 min. After cooling to room temperature, 20 mldiethylether was added. The resulting precipitate (mixture of thediastereomers 650 Compound 1 (isomer 1) and 650 Compound 1 (isomer 2))was extracted by suction, washed with ether, and dried.

The residue was purified by column chromatography (RP-18,acetonitrile/water and concentrated HCl) to separate the diastereomersfrom each other. The diastereomer that first eluted from the column wastermed diastereomer 1 (650 Compound 1 (isomer 1)). The diastereomer thateluted second from the column was termed diastereomer 2 (650 Compound 1(isomer 2)). The diastereomers were separated, followed byneutralization and evaporation. Purification of the singlediastereomeric compound was completed on a RP-18 column,acetonitrile/water. The corresponding fractions were pooled and thesolvent was removed by distillation. The two products (diastereomers 650Compound 1 (isomer 1) and 650 Compound 1 (isomer 2)) were dried in highvacuum.

650 Compound 1 (Isomer 1):

yield: 11%

UV-vis (PBS): λmax=654 nm, λem=672 nm

MS (ESI−) [M/z]: 299.7 [M]³⁻; 461.0 [M+Na]²⁻

650 Compound 1 (Isomer 2):

yield: 24%

UV-vis (PBS): λmax=654 nm, λem=672 nm

MS (ESI−) [M/z]: 299.6 [M]³⁻; 461.1 [M+Na]²⁻

EXAMPLE 14 650 Compound 2 Synthesis of2-{(1E,3E)-5-[1-(5-carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-penta-1,3-dienyl}-1-[2-(2-methoxy-ethoxy)-ethyl]-3-methyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumtri sodium salt

Both 564 mg (1 mmol)1-(5-carboxypentyl)-3-methyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 449 mg (1 mmol)1-[2-(2-methoxy-ethoxy)-ethyl]-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumwere dissolved in 20 ml of acetic acid/acetic anhydride (1/1) followedby the addition of 200 mg of sodium acetate. The synthesis and work-upwere carried out according to Example 13.

650-1 Compound 2:

yield: 11%

UV-vis (PBS): λmax=654 nm, λem=672 nm

MS (ESI−) [M/z]: 314.4 [M]³⁻; 483.0 [M+Na]²⁻

650-2 Compound 2:

yield: 16%

UV-vis (PBS): λmax=654 nm, λem=672 nm

MS (ESI−) [M/z]: 314.5 [M]³⁻; 483.1 [M+Na]²⁻

EXAMPLE 15 650 Compound 3 Synthesis of2-{(1E,3E)-5-[1-(5-carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-penta-1,3-dienyl}-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-3-methyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumtri sodium salt—650 compound 3

Both 564 mg (1 mmol)1-(5-carboxypentyl)-3-methyl-2-((1E,3E)-4-phenylamino-buta-1,3-dienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 493 mg (1 mmol)1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumwere dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed bythe addition of 200 mg sodium acetate. The synthesis and work-up werecarried out according to Example 13.

650-1 Compound 3:

yield: 10%

UV-vis (PBS): λmax=654 nm, λem=672 nm

MS (ESI−) [M/z]: 329.2 [M]³⁻; 505.0 [M+Na]²⁻

650-2 Compound 3:

yield: 23%

UV-vis (PBS): λmax=654 nm, λem=672 nm

MS (ESI−) [M/z]: 329.1 [M]³⁻; 505.1 [M+Na]²⁻

EXAMPLE 16 Synthesis of 755 Compound 12-{(1E,3E,5E)-7-[1-(5-Carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-hepta-1,3,5-trienyl}-1-(2-methoxy-ethyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumtri sodium salt

Six hundred and sixteen mg (1 mmol)1-(5-Carboxypentyl)-3-methyl-2-((1E,3E,5E)-6-phenylamino-hexa-1,3,5-trienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 404 mg (1 mmol)1-(2-methoxy-ethyl)-2,3-dimethyl-5-sulfo-3-(3-sulfopropyl)-3H-indoliumwere dissolved in 20 ml of acetic acid/acetic anhydride (1/1) followedby the addition of 200 mg of sodium acetate. The solution was stirredunder reflux for 15 min. After cooling to room temperature, 20 mldiethylether was added. The resulting precipitate (mixture of thediastereomers 755 Compound 1 (isomer 1) and 755 Compound 1 (isomer 2))was extracted by suction, washed with ether, and dried.

The residue was purified by column chromatography (RP-18,acetonitrile/water and concentrated HCl) to separate the diastereomersfrom each other. The diastereomer that first eluted from the column wastermed diastereomer 1 (755 Compound 1 (isomer 1)). The diastereomer thateluted second from the column was termed diastereomer 2 (755 Compound 1(isomer 2)). The diastereomers were separated, followed byneutralization and evaporation. Purification of the singlediastereomeric compound was completed on a RP-18 column,acetonitrile/water. The corresponding fractions were pooled and thesolvent was removed by distillation. The two products (diastereomers 755Compound 1 (isomer 1) and 755 Compound 1 (isomer 2)) were dried in highvacuum.

755 Compound 1 (Isomer 1):

yield: 8%

UV-vis (PBS): λ_(max)=752 nm; λ_(em)=778 nm

MS (ESI−) [M/z]: 308.4 [M]³⁻; 474.2 [M+Na]²⁻

755 Compound 1 (Isomer 2):

yield: 16%

UV-vis (PBS): λ_(max)=752 nm; λ_(em)=778 nm

MS (ESI−) [M/z]: 308.4 [M]³⁻; 474.2 [M+Na]²⁻.

EXAMPLE 17 Synthesis of 755 Compound 22-{(1E,3E,5E)-7-[1-(5-Carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-hepta-1,3,5-trienyl}-1-(2-methoxy-ethoxy)-3-methyl-5-sulfo-3-(3-sulfopropyl)-3H-indolium

Both 1 mmol1-(5-Carboxypentyl)-3-methyl-2-((1E,3E,5E)-6-phenylamino-hexa-1,3,5-trienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 1 mmol1-[2-(2-methoxy-ethoxy)-ethyl]-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumwere dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed bythe addition of 200 mg sodium acetate. The solution was stirred underreflux for 15 min. After cooling to room temperature, 20 ml diethyletherwas added. The resulting precipitate (mixture of the diastereomers 755compound 2 (isomer 1) and 755 compound 2 (isomer 2)) was extracted bysuction, washed with ether and dried. The residue is purified by columnchromatography (RP-18, acetonitrile/water and concentrated HCl), therebyseparating the diastereomers from each other, as described in Example16.

EXAMPLE 18 Synthesis of 755 Compound 32-{(1E,3E,5E)-7-[1-(5-Carboxypentyl)-3-methyl-5-sulfo-3-(3-sulfopropyl)-1,3-dihydro-indol-(2E)-ylidene]-hepta-1,3,5-trienyl}-1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-3-methyl-5-sulfo-3-(3-sulfopropyl)-3H-indolium

Both 1 mmol1-(5-carboxypentyl)-3-methyl-2-((1E,3E,5E)-6-phenylamino-hexa-1,3,5-trienyl)-5-sulfo-3-(3-sulfopropyl)-3H-indoliumand 1 mmol1-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl}-2,3-dimethyl-5-sulfo-3-(3-sulfo-propyl)-3H-indoliumwere dissolved in 20 ml acetic acid/acetic anhydride (1/1) followed bythe addition of 200 mg sodium acetate. The solution was stirred underreflux for 15 min. After cooling to room temperature, 20 ml diethyletherwas added. The resulting precipitate (mixture of the diastereomers 755compound 3 (isomer 1) and 755 compound 3 (isomer 2)) was extracted bysuction, washed with ether and dried. The residue is purified by columnchromatography (RP-18, acetonitrile/water and concentrated HCl), therebyseparating the diastereomers from each other, as described in Example16.

EXAMPLE 19

Properties of 650 Compounds as NHS esters were compared withcommercially available dyes, as shown below.

650 1/1-NHS 650 1/1-NHS Alexa Fluor CF 647- (tetrasulfonated) 6504/4-NHS (disulfonated) 647-NHS NHS MW (g/mol) 1066 1424.63 789.91 ~11503241 Ex (nm) 652 658 651 650 650 Em (nm) 672 681 665 665 665 ε (M⁻¹cm⁻¹)250,000 250,000 250,000 239,000 240,000 (theoretical) PEG 1/1 4/4 1/1N/A unknown (length/# of chain) Sulfonate 4 2 2 unknown

Properties of 755 Compounds as NHS esters were compared withcommercially available dyes as shown below:

DyLight 800-NHS 755 Compound 4/4-NHS MW (g/mol) 1050 1684.9 Ex (nm) 777783 Em (nm) 794 797 ε (M⁻¹cm⁻¹) 270,000 270,000 (theoretical) PEG N/A4/4 (length/number of PEG groups on compound Sulfonates 3 3

Excitation/emission spectra of 755 Compound 4/4-NHS was within +/−10 nmcompared to DyLight 800-NHS.

EXAMPLE 20

DyLight 650 1/1 and DyLight 650 4/4 were dissolved in dimethylformamide(DMF) at 10 mg/ml, mixed on a vortex mixer for 15 seconds, and observedto determine if the dyes went into solution. The dyes were then allowedto incubate for five minutes and again mixed on a vortex mixer for 30seconds. DyLight 650 (4/4) dissolved immediately and DyLight 650 (1/1)did not go into solution until it was incubated for five minutes andmixed again.

EXAMPLE 21

Inventive and commercial compounds, each as the NHS ester, wereconjugated to goat anti-mouse (GAM) and goat anti-rabbit (GAR)antibodies. GAM and GAR at 10 mg/ml in phosphate buffered saline (PBS),were dialyzed against 50 mM borate buffer, pH 8.5. The compounds werereconstituted in DMF, and CF 647 was reconstituted in dimethylsulfoxide(DMSO), at 10 mg/ml and combined at 2.5×, 5×, 10×, or 15× molar excesswith GAM or GAR for about two hours at room temperature to label theantibodies.

Labeled compounds, also termed dyes or labels, were subjected to PDDR toremove the unlabeled (free) compound; 100-200 μl of the packed resin wasused per mg of protein purified. The purified antibody-labeled dyes werethen diluted 1:50 in PBS and scanned for absorbance from 700 nm to 230nm on a UV Cary spectrophotometer to determine the proteinconcentration, and to determine the mole dye to mole protein ratio. Eachconjugate was diluted 1:10 in 50% glycerol and heated in the presence of10 mM dithiothreitol (DTT) for 5 min at 95° C., then separated byelectrophoresis on polyacrylamide gels in the presence of sodium dodecylsulfate (SDS-PAGE). The gels were scanned using the Typhoon 9400 Imagerto verify removal of the unconjugated compound. Labeling efficiency wascompared, with results showing degree of labeling below, where 650Compound 1/1 (4S) denotes four sulfo groups on the compound and 650Compound 1/1 (2S) denotes two sulfo groups on the compound.

GAM 2.5X 5X 10X 15X 650 Compound 4/4-NHS 2.4 4.7 9.6 14.2 650 Compound1/1 (4S)-NHS 2.6 4.4 7.9 10.0 CF 647-NHS 1.7 2.9 4.3 5.1 Alexa Fluor647-NHS 2.4 4.0 6.5 8.2

GAR 2.5X 5X 10X 15X 650 Compound 4/4-NHS 2.3 4.6 8.5 13.4 650 Compound1/1 (4S)-NHS 2.8 4.1 7.6 10.0 CF 647-NHS 1.7 2.6 4.2 4.9 Alexa Fluor647-NHS 2.3 3.6 6.1 7.7Labeling efficiency was the highest for all 650 Compound 4/4 conjugates,followed by 650 Compound 1/1, compared to the other dyes at each molarexcess. 650 Compound 1/1 and CF 647 required extra time for completesolubility.

755 Compound 4/4 and DyLight 800, each as the NHS ester, were conjugatedto goat anti-mouse (GAM) and goat anti-rabbit (GAR) antibodies asdescribed above. Briefly, 755 Compound 4/4-NHS and DyLight 800-NHS werereconstituted at 10 mg/ml in DMF. Each compound showed good solubility.One mg in 100 μl of GAM or GAR was prepared at 10.0 mg/ml in boratebuffer pH 8.5. Ten mg in 1000 μl of GAM was prepared at 10.0 mg/rd inborate buffer pH 8.5. Ten mg in 1000 μl of GAM was prepared at 10.0mg/ml in PBS pH 7.4. GAM and GAR were labeled with each compound at2.5×, 5×, 7× (10 mg) and 9× molar excesses. Ten mg GAM was labeled witheach compound at 5× and 7× molar excesses in borate buffer. Ten mg GAMwas labeled with each compound at 5× and 7× molar excesses in PBSbuffer, and incubated for greater than 60 min. Conjugates were purifiedon Pierce Dye Removal Resin in Harvard columns with 100-200 μl resin permg of each conjugate. All the conjugates were diluted 1:50 and scannedon UV Cary Spectrophotometer. Absorption maxima for free compounds isshown in FIG. 1A, where free dye 755 Compound 4/4-NHS showed a singleabsorption peak at 783 nm and free Dye DyLight 800-NHS showed a singleabsorption peak at 770 nm. Absorption maxima of GAM conjugates with 755Compound 4/4 (D/P of 2.2) and DyLight 800 (D/P of 1.4), are shown inFIG. 1B, with baseline 100% transmission shown. 755 Compound 4/4-GAMconjugates showed a single absorption peak at 783 nm and DyLight 800-GAMconjugates showed 2 peaks: major at 775 nm and minor at 706 nm.Absorption maxima of GAR conjugates with 755 Compound 4/4 (D/P of 2.2),DyLight 800 (D/P of 1.4), and IR800-GAR (D/P of 2.6; conjugate madeusing LI-COR IR800 Dye) are shown in FIG. 1C, with baseline 100%transmission shown. 755 Compound 4/4-GAR conjugates showed a singleabsorption peak at 783 nm, DyLight 800-GAR conjugates showed 2absorption peaks: major at 774 nm and minor at 705 nm, and IR800-GARshowed a main peak at 779 nm and a minor peak at 709 nm. The secondarypeak observed with Daylight 800 at 706 nm is approximately 60% of themain peak at 775 nm, and this secondary peak can affect the readoutdepending on the instrumentation used.

Labeling efficiency was compared, with results showing degree oflabeling (D/P) below.

2.5X 5X 7X 9X GAM borate buffer 755 Compound 4/4-NHS 0.97 1.72 2.24 2.94DyLight 800-NHS 0.74 1.12 1.41 1.91 GAR borate buffer 755 Compound4/4-NHS 0.84 1.64 2.22 2.91 DyLight 800-NHS 0.71 1.17 1.39 1.88 GAMborate buffer 755 Compound 4/4-NHS 1.75 2.32 DyLight 800-NHS 1.20 1.55GAM PBS buffer 755 Compound 4/4-NHS 0.74 1.16 DyLight 800-NHS 0.89 1.15

Conjugation of 755 Compound 4/4 in borate buffer resulted in about 50%higher labeling efficiency than DyLight 800 at similar molar excess. Forboth dyes, conjugations performed in PBS buffer pH 7.2 showed about twotimes less labeling efficiency compared to conjugations in borate bufferpH 8.5. DyLight 800 conjugates at 7× molar excess in borate bufferprecipitated within three days and 755 Compound 4/4 conjugates had novisible precipitation after three weeks. Use of 755 Compound 4/4 at 5×molar excess avoids dye aggregation and yields a similar D/P as DyLight800 at 7× molar excess.

EXAMPLE 22

Performance of the dye-GAM conjugates and dye-GAR conjugates wasevaluated in a functional assay. Wells of a 96 white opaque plate werecoated with target proteins mouse IgG immunoglobulin or rabbit IgGimmunoglobulin. One hundred μl mouse or rabbit IgG at a concentration of10 μg/ml was applied to the corresponding wells in columns 1 and 2. Thetarget proteins were serially diluted 1:1 from the wells in columns 2 to11 using 100 μl PBS. One hundred μl of the samples from the wells incolumn 11 were discarded. One hundred μl PBS was added to the wells incolumn 12. The plates were incubated overnight at 4° C. and then blocked2×200 μl with Thermo Scientific SuperBlock® Blocking Buffer. The coatedplates were washed 2×200 μl with PBS-Tween and 1×200 μl with PBS. Basedon the calculated concentrations, GAM and GAR conjugates were diluted1:250 (of 1 mg/ml) in PBS buffer. Conjugates diluted in PBS to 4 μg/mlwere added to the corresponding plates (100 μl/well) and then incubatedfor 1 h in the dark. The plates were washed with 2×200 μl with PBS-Tweenand 1×200 μl with PBS and filled with PBS buffer (100 μl/well) prior toscanning on Tecan Safire to detect fluorescence intensity.

As shown in FIGS. 2-9, the relative fluorescence units (RFU) orsignal-to-background ratio (S/B) of the dyes were compared at variousconcentrations, using the indicated conjugation conditions.

FIG. 2 shows results of a functional assay using GAR conjugated witheither 650 Compound 4/4 (diamonds), 650 Compound 1/1 (circles), CF 647(triangles), or Alexa Fluor 647 (squares) at a 2.5× molar excess of thedyes, showing relative fluorescence units (RFU) versus amount of targetantibody per well (ng/well). FIG. 3 shows the signal-to-background ratio(SB) of the functional assay of FIG. 2, showing either 650 Compound 4/4(diamonds), 650 Compound 1/1 (circles), CF 647 (triangles), or AlexaFluor 647 (squares) at a 2.5× molar excess of the dyes. FIG. 4 showsresults of a functional assay using GAR conjugated with either 650Compound 4/4 (diamonds), 650 Compound 1/1 (circles), CF 647 (triangles),or Alexa Fluor 647 (squares) at a 5× molar excess of the dyes, showingrelative fluorescence units (RFU) versus amount of target antibody perwell (ng/well). FIG. 5 shows the signal-to-background ratio (SB) of thefunctional assay of FIG. 4, showing either 650 Compound 4/4 (diamonds),650 Compound 1/1 (circles), CF 647 (triangles), or Alexa Fluor 647(squares) at a 5× molar excess of the dyes. 650 Compound 1/1-GAR was thebest performing conjugate at all molar excesses. Up to 125 ng/well ofrabbit IgG, the 650 Compound 4/4-GAR showed similar binding fluorescenceas Alexa Fluor 647-GAR but better than CF 647-GAR. CF 647 showedsignificantly lower performance of all the conjugates.

FIG. 6 shows results of a functional assay using GAM conjugated witheither 650 Compound 4/4 (diamonds), 650 Compound 1/1 (circles), CF 647(triangles), or Alexa Fluor 647 (squares) at a 2.5× molar excess of thedyes, showing relative fluorescence units (RFU) versus amount of targetantibody per well (ng/well). FIG. 7 shows the signal-to-background ratio(SB) of the functional assay of FIG. 6, showing either 650 Compound 4/4(diamonds), 650 Compound 1/1 (circles), CF 647 (triangles), or AlexaFluor 647 (squares) at a 2.5× molar excess of the dyes. FIG. 8 showsresults of a functional assay using GAM conjugated with either 650Compound 4/4 (diamonds), 650 Compound 1/1 (circles), CF 647 (triangles),or Alexa Fluor 647 (squares) at a 5× molar excess of the dyes, showingrelative fluorescence units (RFU) versus amount of target antibody perwell (ng/well). FIG. 9 shows the signal-to-background ratio (SB) of thefunctional assay of FIG. 8, showing either 650 Compound 4/4 (diamonds),650 Compound 1/1 (circles), CF 647 (triangles), or Alexa Fluor 647(squares) at a 5× molar excess of the dyes. 650 Compound 1/1-GAM was thebest performing conjugate at all molar excesses. Up to 125 ng/well ofmouse IgG, 650 Compound 4/4-GAM showed similar binding fluorescence asAlexa Fluor 647-GAM but better than CF 647-GAM. CF-647 showedsignificantly lower performance of all the conjugates.

Performance of the dye-GAM conjugates and dye-GAR conjugates wasevaluated in a functional fluorescence plate assay, as described above.After incubation, the plates were scanned on LiCor Odyssey at 800channel (unbound). Plates were washed 2×200 μl with PBS-Tween20 and1×200 μl with PBS and filled with PBS buffer (100 μl/well) prior toscanning on LiCor Odyssey at 800 channel (bound). Average total unboundfluorescence intensity is shown in FIG. 10A, and the corresponding plateimage in FIG. 10B, showing 1 (755 Compound 4/4-GAR; 2.5× molar excess;D/P 0.84), 2 (755 Compound 4/4-GAR; 5× molar excess; D/P 1.64), 3 (755Compound 4/4-GAR; 7× molar excess; D/P 2.22), 4 (755 Compound 4/4-GAR;9× molar excess; D/P 2.91), 5 (DyLight 800-GAR; 2.5× molar excess; D/P0.71), 6 (DyLight 800-GAR; 5× molar excess; D/P 1.17), 7 (DyLight800-GAR; 7× molar excess; D/P 1.39), and 8 (DyLight 800-GAR; 9× molarexcess; D/P 1.88). Average total bound fluorescence intensity is shownin FIG. 11A, with 755 Compound 4/4-GAR (solid lines) and DyLight 800-GAR(dotted lines) at either 2.5× molar excess (triangles), 5× molar excess(diamonds), 7× molar excess (circles), or 9× molar excess (squares).FIG. 11B shows the plate image of FIG. 11A, showing 1 (755 Compound4/4-GAR; 2.5× molar excess), 2 (755 Compound 4/4-GAR; 5× molar excess),3 (755 Compound 4/4-GAR; 7× molar excess), 4 (755 Compound 4/4-GAR; 9×molar excess), 5 (DyLight 800-GAR; 2.5× molar excess), 6 (DyLight800-GAR; 5× molar excess), 7 (DyLight 800-GAR; 7× molar excess), and 8(DyLight 800-GAR; 9× molar excess). In the above functional fluorescenceplate assays, 755 Compound 4/4-GAR showed significantly higher bindingfluorescence intensity compared to DyLight 800-GAR conjugates in boratebuffer.

Average total unbound fluorescence intensity is shown in FIG. 12A, andcorresponding plate image in FIG. 12B, showing 1 (755 Compound 4/4-GAR;7× molar excess; D/P 2.22), 2 (DyLight 800-GAR; 7× molar excess; D/P1.39), 3 (IR800-GAR (Rockland); D/P 2.6), and 4 (DyLight 800-GAR(Rockland); D/P 2.1). Average total bound fluorescence intensity isshown in FIG. 13A, with 755 Compound 4/4-GAR (diamonds; 7× molar excess;D/P 2.24), DyLight 800-GAR (circles; 7× molar excess; D/P 1.39),IR800-GAR (Rockland) (triangles; D/P 2.6), and DyLight 800-GAR(Rockland) (squares; D/P 2.1). FIG. 13B shows the plate image of FIG.13A, showing 1 (755 Compound 4/4-GAR; 7× molar excess), 2 (DyLight800-GAR; 7× molar excess), 3 (IR800-GAR (Rockland)), and 4 (DyLight800-GAR (Rockland)). In the above functional fluorescence plate assays,755 Compound 4/4-GAR showed significantly higher binding fluorescenceintensity compared to DyLight 800-GAR conjugates in borate buffer. Atsimilar D/P of about 2, 755 Compound 4/4-GAR performed better thanIR800-GAR (conjugate made by Rockland using IR800 Dye from LiCOR; soldin lyophilized form) and DyLight 800-GAR (conjugate made by Rocklandusing DyLight 800; sold in lyophilized form). No quenching at high dyemolar excesses was observed with 755 Compound 4/4.

EXAMPLE 23

The inventive compounds and commercial dye were evaluated forimmunofluorescence in cell based assays using the following protocol.Plates containing U2OS cells (human osteosarcoma cell line) were fixedin 4% paraformaldehyde in PBS/0.1% Triton X-100 for 15 min at roomtemperature. The cells were then permeabilized with 2% BSA in PBS/0.1%Triton X-100 for 15 min at room temperature. Negative controls containonly 2% BSA/PBS-0.1% Triton-X100 blocker. Diluted primary antibodies,mouse-anti-protein disulphide isomerase (PDI) or rabbit-anti-HDAC2,diluted in 2% BSA/PBS-0.1% Triton-X100 were added to the plates andincubated for one hour at room temperature. Negative controls containonly 2% BSA/PBS-0.1% Triton-X100 blocker. The plates were washed 3×100μl with PBS. Based on the calculated protein concentrations, theconjugates made in Example 22 were diluted to 4 μg/ml in PBS/0.1% TritonX-100 and added to the plates (50 μl/well) and incubated one hour in thedark at room temperature. After incubation, the primary antibodysolution was removed from the plates, and the plates were washed 3×100μl with PBS. One hundred μl of 0.1 μg/ml Hoechst dye in PBS was addedper well. The plates were then scanned on an ArrayScan® Plate Reader forimaging and quantitation.

FIGS. 14A-E shows results of an immunofluorescence assay usingrabbit-anti-HDAC2 as a primary antibody, and either 650 Compound 4/4-GAR(FIG. 14A; column A), 650 Compound 1/1 (4S)-GAR (FIG. 14B; column A), CF647-GAR (FIG. 14C; column A), Alexa Fluor 647-GAR (FIG. 14D; column A),or 650 Compound 1/1 (2S)-GAR (FIG. 14E; column A) as secondary antibody,with negative controls shown in column B, where the compound wasconjugated to GAR (secondary antibody) at 2.5× molar excess (row 1), 5×molar excess (row 2), 10× molar excess (row 3), or 15× molar excess (row4). Non-specific binding was observed with all the conjugates at highdye molar excesses. 650 Compound 4/4-GAR was not as bright as 650Compound 1/1-GAR at the lower molar excesses 2.5× and 5×. However, 650Compound 4/4-GAR did not appear to quench, which was observed with 650Compound 1/1-GAR. CF 647-GAR and Alexa Fluor 647-GAR conjugates showedlower intensity at all molar excesses.

Quantitative analysis of the data of FIGS. 14A-E, expressed as MeanTotal Intensity, which is the average total intensity of all pixelswithin a defined area or defined primary object such as a nucleus, isshown in FIG. 15, and signal to background ratio (S/B) is shown in FIG.16, at molar excesses of 2.5× (open bars), 5× (upward diagonal linedbars), 10× (downward diagonal lined bars), and 15× (vertical linedbars). 650 Compound 1/1-GAR and 650 Compound 4/4-GAR showed the highestfluorescence intensity. While 650 Compound 4/4-GAR does not appear toquench, 650 Compound 1/1, CF 647, and Alexa Fluor 647 conjugates showedsignificant quenching at high molar excesses. CF 647-GAR and Alexa647-GAR conjugates showed lower intensity at all molar excesses.

FIGS. 17A-D show immunofluorescence assay results using mouse-anti-PDIas a primary antibody, and either 650 Compound 4/4-GAR (FIG. 17A; columnA), 650 Compound 1/1 (4S)-GAR (FIG. 17B; column A), CF 647-GAR (FIG.17C; column A), or Alexa Fluor 647-GAR (FIG. 17D; column A) as secondaryantibody, with negative controls shown in column B, where the compoundwas conjugated to GAM (secondary antibody) at 2.5× molar excess (row 1),5× molar excess (row 2), 10× molar excess (row 3), or 15× molar excess(row 4). No non-specific binding was observed with any the dyes(purified with 200 μl of resin/mg of protein). 650 Compound 4/4-GAM wasthe brightest at all molar excesses. 650 Compound 1/1, CF 647, and AlexaFluor 647 conjugates showed significant quenching above 2.5× molarexcess. CF 647-GAM and Alexa 647-GAM conjugates showed lower intensityat all molar excesses.

Quantitative analysis of the data of FIGS. 17A-D, expressed as MeanTotal Intensity, which is the average total intensity of all pixelswithin a defined area or defined primary object such as a nucleus, isshown in FIG. 18 and signal to background ratio (S/B) is shown in FIG.19, at molar excesses of 2.5× (open bars), 5× (upward diagonal linedbars), 10× (downward diagonal lined bars), and 15× (vertical linedbars). 650 Compound 1/1, CF 647, and Alexa Fluor 647 conjugates showingtheir highest binding intensity at 2.5× dye molar excess. 650 Compound4/4-GAM was the brightest at all molar excesses. 650 Compound 4/4-GAMshowed quenching above 5× molar excess, while 650 Compound 1/1, CF 647,and Alexa Fluor 647 conjugates showed significant quenching above 2.5×molar excess. CF 647-GAM and Alexa 647-GAM conjugates showed lowerintensity at all molar excesses.

FIGS. 20A-D shows results of an immunofluorescence assay usingrabbit-anti-HDAC2 as a primary antibody, and either 650 Compound 4/4-GAR(FIG. 20A; column A), 650 Compound 1/1 (4S)-GAR (FIG. 20B; column A), CF647-GAR (FIG. 20C; column A), or Alexa Fluor 647-GAR (FIG. 20D; columnA) as secondary antibody, with negative controls shown in column B,where the compound was conjugated to GAR (secondary antibody) at 2.5×molar excess (row 1), 5× molar excess (row 2), 10× molar excess (row 3),or 15× molar excess (row 4). No non-specific binding was observed withany of the conjugates (purified with 200 μl of resin/mg of protein). 650Compound 4/4-GAR was the brightest at all molar excesses. 650 Compound1/1, CF 647, and Alexa Fluor 647 conjugates showed significant quenchingabove 2.5× molar excess. CF 647-GAR and Alexa 647-GAR conjugates showedlower intensity at all molar excesses.

Quantitative analysis of the data of FIGS. 20A-D, expressed as MeanTotal Intensity, which is the average total intensity of all pixelswithin a defined area or defined primary object such as a nucleus, isshown in FIG. 21 and signal to background ratio (S/B) is shown in FIG.22, at molar excesses of 2.5× (open bars), 5× (upward diagonal linedbars), 10× (downward diagonal lined bars), and 15× (vertical linedbars). 650 Compound 1/1, CF 647, and Alexa Fluor 647 conjugates showedthe highest binding intensity at 2.5× dye molar excess, however 650Compound 4/4 was the brightest at all molar excesses. While 650 Compound4/4-GAR did not appear to quench, 650 Compound 1/1, CF 647, and AlexaFluor 647 conjugates showed significant quenching above 2.5× molarexcess. CF 647-GAR and Alexa 647-GAR conjugates showed lower intensityat all molar excesses.

The data indicated that excitation/emission spectra of 650 Compound4/4-NHS was within +/−10 nm compared to 650 Compound 1/1-NHS, AlexaFluor 647-NHS, and CF 647-NHS ester. Labeling efficiency of 650 Compound4/4 was the highest, followed by 650 Compound 1/1, compared to the otherdyes at each molar excess. 650 Compound 1/1 and CF 647 required extratime for complete solubility. 650 Compound 1/1-GAM/R was the bestperforming conjugate at all molar excesses. Up to 125 ng/well ofmouse/rabbit IgG, 650 Compound 4/4-GAM showed similar bindingfluorescence as 650 Compound 1/1-GAM and Alexa Fluor 647-GAM, but betterthan CF 647-GAM. CF 647 showed significantly lower performance of alldyes. When purified with 200 μl of resin/mg of protein, the conjugatesdid not show any non-specific binding. Conjugates made with 650 Compound4/4 (GAM/R) were the brightest and showed the highest signal tobackground ratios. 650 Compound 1/1, CF 647, and Alexa Fluor 647conjugates showed significant quenching above 2.5× molar excess. CF 647and Alexa Flour 647 conjugates showed lower intensity at all molarexcesses.

EXAMPLE 24

The inventive compounds are evaluated for stability. All compounds arepacked under argon in plastic vials. The vials are sealed with a dryingpad in an aluminium coated pouch, and then stored at 50° C. for sevendays.

EXAMPLE 25

The inventive compounds are evaluated in direct fluorescence labeling ofcell surface proteins using methods known in the art. For example,suitable cell plates, such as IMR90 cells (human lung embryonicfibroblast), are washed and then incubated with the conjugates. The cellplates are then washed and imaged using an appropriate instrument, suchas a Thermo Scientific ArrayScan VTI HCS Reader.

EXAMPLE 26

The inventive compounds are used for in vivo imaging to obtaininformation about biological tissues that are not readily accessible.The compounds are responsive to light in the near infrared (NIR) regionof the spectrum, which is a part of the spectrum that has minimalinterference from the absorbance of biological materials. In oneembodiment, the compounds are used for fluorescent imaging of targetswithin animals. For example, in vivo imaging information can be obtainedusing methods such as X-ray, magnetic resonance imaging, positronemission tomography, ultrasound imaging and probing, and othernon-invasive methods used for diagnosing and treating disease. Light inthe NIR region, from about 650 nm to about 1000 nm wavelength, canpermeate through several centimeters of tissue and therefore, can beused for in vivo imaging. Fluorescent dyes, such as the inventivecompounds that are responsive to light in these longer wavelengths, canbe used as conjugates with targeting molecules such as antibodies tobind and accumulate in, e.g., diseased tissue such as tumors, and may beused to distinguish healthy from diseased tissue. In some methods, theinventive compound may be attached to a biomolecule, such as a protein,peptide, or a drug, which is localized or retained in the desired tissueenvironment. Fluorescent in vivo imaging using NIR dyes such as theinventive compounds are diagnostic agents to discretely target diseasetissue directly within animals or humans.

For in vivo imaging, the compound or a conjugate of the compound with atargeting agent, is administered to a tissue (e.g., intravenously),permitted to accumulate with excess compound removed by the circulatorysystem, then the tissue is irradiated with light at an appropriatewavelength. NIR fluorescent light is recorded and/or an image isgenerated from the data obtained to specifically detect and visualizethe targeted cells or tissues. The dose of compound administered candiffer depending upon the specific tissue, application, etc., as long asthe method achieves a detectable concentration of the compound in thetissue to be assessed.

EXAMPLE 27

The inventive dyes were evaluated in biodistribution and bioclearancestudies. One mg NHS-DyLight 650 4/4 and NHS-DyLight 650 1/1 arereconstituted to 10 mg/ml and diluted to 1 mg/ml in PBS. The dyes areincubated for 30 minutes and then quenched by adding one-tenth volume of3M N-ethanolamine. One hundred μL of 1 mg/mL of each hydrolyzed dyesolution is intravensouly injected via the retro orbital plexus ofnon-tumored nude mice. One mouse is injected for each dye. The animalsare imaged on a Carestream MSFX at 0 h, 3 h, 6 h, 12 h, and 24 h postinjection. After the final time point, animals are sacrificed andtissues collected for ex vivo imaging. The heart, liver, spleen, lungs,and kidneys are gathered from one mouse from each cohort, and fixed andstained using hematoxalin and eosin. Colorimetric images are acquired at20× on a Nikon 90i microscope. The results show that the hydrolyzedNHS-DyLight 650 4/4 is removed from the circulatory system, whereashydrolyzed NHS-DyLight 650 1/1 NHS-Alexa 647 accumulate in the liver.

EXAMPLE 28 In Vivo Imaging Using 650 Compound 4/4 Conjugated toAnti-HER2 Antibody

650 Compound 4/4-NHS is conjugated to a rabbit anti-HER2 antibody(Genscript USA, Piscataway N.J.) by reconstituting the compound indimethylformamide (DMF) at 10 mg/ml, then incubated at 10× molar excesswith rabbit anti-HER2 antibody (0.1 mg) for 1 h at room temperature toresult in a 650 Compound 4/4-anti-HER2 conjugate. The sample is thensubjected to PDDR to remove unlabeled (free) 650 Compound 4/4. Tenmicrogram of the conjugate is injected intravenously (IV) to athymicmice bearing BT474 tumors. The animals are imaged over time at 1, 24,48, 72, 96, and 120 hours post-injection using Pearl Impulse Imager fromLI-COR Biosciences (LI-COR Instruments, Lincoln Nebr.).

Upon whole body imaging, fluorescence intensity is observed to bedistributed over the whole animal during the first hour imagining anddiminishes significantly at 72 hours. After 96 hours, the signal islocalized and specific to the tumor.

EXAMPLE 29 In Vivo Imaging Using Either Monosulfonated or Disulfonated650 Compound 4/4

The compound may be rendered less hydrophilic, i.e., more hydrophobic orless negatively charged, by altering the number of sulfonate groups.Fewer sulfonates render the compound more hydrophobic and lessnegatively charged. In this embodiment, the compound may be more readilyretained in a desired tissue or location if the appropriate number ofsulfonates is determined. For example, compound penetration into cellsis more efficient if fewer sulfonates are on the compound. The compoundmay contain one, two, three, or four sulfonate groups. Hydrophobiccompounds are also known to more efficiently cross the cell membrane,and therefore are more desirable when the target of interest is locatedwithin the cell.

Alendronate, a compound that binds to, and is retained in, LNCapprostate cancer cells, is conjugated with disulfonated or monosulfonated650 Compound 4/4 by incubating a solution containing 1 mM disulfonatedor monosulfonated 650 Compound 4/4-NHS in 1 ml of PBS and 0.5 mltetrahydrofuran (THF) with 0.1 mM alendronate and 0.2 mMdiisopropylethylamine at room temperature overnight. The conjugate ispurified using reverse phase HPLC with 0-50% methanol against a 0.1 Mammonium acetate buffer, and is then lyophilized.

LNCap cells are grown orthotopically in nude mice. 650 Compound 1(isomer 1)-alendronate (5 nmole) is injected into the tumor. Controlmice are injected with free 650 Compound 4/4 containing a carboxylicacid residue instead of the reactive NHS ester. X-ray and near infra-redfluorescence images are captured.

Upon imaging the whole mouse, both the monosulfonated and disulfonated650 Compound 4/4-alendroneate conjugate is retained in mouse tissue butthe free dyes are not retained; the conjugate is retained in the LNCapcell-induced tumor for at least 18 hrs.

EXAMPLE 30 In Vivo Imaging Using Either Monosulfonated or Disulfonated650 Compound 4/4

A drug delivery nanoparticle system conjugated with disulfonated andmonosulfonated 650 Compound 4/4 is prepared as follows. A solutioncontaining 1 mM disulfonated or monosulfonated 650 Compound 4/4-NHS in 1ml of PBS is incubated overnight at room temperature with 0.1 mM of ananti-cancer drug conjugated with transferrin in the form of ananoparticle. The resulting 650 Compound 4/4-nanoparticle conjugates arepurified by centrifugation, and then lyophilized.

The 650 Compound 4/4-nanoparticle conjugates (1 nmole) are injectedintravenously into the tail vein of different mice. Control mice areinjected with non-reactive 650 Compound 4/4 dye containing a carboxylicacid residue instead of a reactive NHS ester. X-ray and near infra-redfluorescence images of mouse brain are captured.

Both 650 Compound 4/4-nanoparticle conjugates are found to localize inthe mouse brain for greater than about 24 hours after injection. Tumorsize progressively decreases after injection of 650 Compound4/4-nanoparticle conjugate, compared to 650 Compound 4/4-nanoparticlewithout the anti-cancer drug.

EXAMPLE 31

The mono-sulfonated derivative could be on any one of six possiblepositions on the 650 compound, accounting for the stereochemistry aroundthe carbon positions on the rings as well as the non-symmetrical natureof the two ends of each dye. Similarly, the di- and tri-substitutedsulfonates could be on multiple possible positions on the inventivecompounds.

EXAMPLE 32

Log P (partition coefficient) and log D (distribution coefficient) ofinventive and commercial compounds were determined to assess compoundhydrophilicity. The log P value of a compound is the logarithm of acompound's partition coefficient between n-octanol and waterlog(C_(octanol)/C_(water)), and is a water, well established measure ofa compound's hydrophilicity. Log P is a constant for the molecule underits neutral form. Low hydrophilicity, and thus high log P, causes poorabsorption or permeation. For compounds to have a reasonable probabilityof being well absorbed, their log P is generally <5.0. Lipophilicity isnot determined by the partitioning of the neutral species inoctanol/water, but by the distribution of both the neutral andpositively charged forms of the molecule. Log D is related tohydrophilicity of a compound. The distribution coefficient, given by logD, takes into account all neutral and charged forms of the molecule.Because the charged forms generally do not enter the octanol phase, thisdistribution varies with pH. In the pH range where the molecule ismostly un-ionized, log D=log P. In the pH range where a significantfraction is ionized, log D becomes a function of log P, pH, and pKa. Ifone assumes that charged molecules do not enter the octanol at all, logD can be expressed as log D=log P−log(1+10**(pH−pKa)).

The following table provides theoretical calculated log D and log Pvalues for 755 Compound 4/4 and DyLight 800 measured by the ChemAxonprogram.

755 Compound 4/4 pH LogD 1.50 −1.43 5.00 −1.77 6.50 −1.77 7.40 −1.77LogP ionic species = −1.8 LogP nonionic species = 1.0 (3.2 consideringtautomerization/resonance) LogD at pl = 0.2 (2.4 consideringtautomerization/resonance)

DyLight 800 pH LogD 1.50 1.44 (1.42 consideringtautomerization/resonance) 5.00 −0.31 6.50 −0.32 7.40 −0.32 LogP ionicspecies = −0.3 LogP nonionic species = 2.4 (4.7 consideringtautomerization/resonance) LogD at pl = 2.8 (5.1 consideringtautomerization/resonance)

The log P and log D calculations comparing 755 Compound 4/4 with fourPEG₄ chains and the DyLight 800 dye from Thermo Fisher with no PEGmodifications illustrates the unexpected benefits that even short PEG₄chains have on the hydrophilicity of cyanine-type dyes. Although theliterature teaches the hydrophilicity benefits of longer PEG polymermodifications on small dye molecules, it does not suggest that suchbenefits occur with short PEG chains. The DyLight 800 dye calculation oflog D indicated that the three sulfonates on the molecule resulted in amildly hydrophilicity index of −0.32 around neutral pH. By contrast, 755Compound 4/4 displayed much better hydrophilicity with a log Ddetermination of −1.77 at the same neutral pH values. This calculateddifference in the hydrophilicity corresponded to observed results thatthe PEGylated dye goes into aqueous solution much more readily that themore hydrophobic DyLight 800 dye. Increased solubility of the PEGylateddye was also seen when dissolving the dyes in a water-miscible organicsolvent such as DMF or DMSO. Conjugates of 755 Compound 4/4 withantibody molecules were much more stable in solution at all levels ofdye-to-protein ratios than the DyLight 800 conjugates. In fact, DyLight800-antibody conjugates tended to precipitate out of solution during theconjugation reaction and had stability issues upon storage, whereas 755Compound 4/4-antibody conjugates did not precipitate during conjugationand did not have storage stability issues. These observations emphasizedthe unexpected benefits the invention provided for cyanine dye compoundsmodified with the relatively short PEG chains described herein.

The embodiments shown and described in the specification are onlyspecific embodiments of inventors who are skilled in the art and are notlimiting in any way. Therefore, various changes, modifications, oralterations to those embodiments may be made without departing from thespirit of the invention in the scope of the following claims. Thereferences cited are expressly incorporated by reference herein in theirentirety.

What is claimed is:
 1. A compound of general formula IIa

or general formula IIb

wherein each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of aliphatic,heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO₃, a PEGgroup P-L-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a (poly)ethylene glycol group where the(poly)ethylene glycol group is (CH₂CH₂O)_(s) where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P-L-Z, and a caboxamidegroup -L-CONH—P-L-Z; each of R⁷ and R⁸ is the same or different and isindependently selected from the group consisting of H, SO₃, a PEG groupP-L-Z where P is selected from an ethylene glycol group, a diethyleneglycol group, and a (poly)ethylene glycol group, where the(poly)ethylene glycol group is (CH₂CH₂O)_(s), where s is an integer from3-6 inclusive, a sulfonamide group —SO₂NH—P-L-Z, and a caboxamide group—CONH—P-L-Z; where L is selected from the group consisting of a divalentlinear (—(CH₂)_(o)—, o=0 to 15), crossed, or cyclic alkane group that isoptionally substituted by at least one atom selected from the groupconsisting of oxygen, substituted nitrogen, and/or sulfur; where Z isselected from the group consisting of H, CH₃, alkyl, heteroalkyl, NH₂,—COO⁻, —COOH, —COSH, CO—NH—NH₂, —COF, —COCl, —COBr, —COI, —COO—Su(succinimidyl/sulfosuccinimidyl), —COO—STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′—CO—CH₂—I, —CONR′R″, —CONR′-biomolecule, —CONR′-L-COO⁻,—CONR′-L-COOH, —CONR′-L-COO—Su, —CONR′-L-COO—STP, —CONR′-L-COO-TFP,—CONR′-L-CONR″₂, —CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂,—CONR′-L-OH, —CONR′-L-O-phosphoramidite, —CONR′-L-CHO,—CONR′-L-maleimide, and —CONR′-L-NH—CO—CH₂—I; R′ and R″ is selected fromthe group consisting of H, aliphatic group, and heteroaliphatic group,and the biomolecule is a protein, antibody, nucleotide, oligonucleotide,biotin, or hapten; X is selected from the group consisting of —SH, —NH₂,—NH—NH₂, —F, —Cl, —Br, I, —NHS (hydroxysuccinimidyl/sulfosuccinimidyl),—O-TFP (2,3,5,6-tetrafluorophenoxy), —O—STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH₂—I, where R is —H or an aliphatic or heteroaliphaticgroup; Kat is a number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s)needed to compensate the negative charge brought by the cyanine; m is aninteger from 0 to 5 inclusive; o is an integer from 0 to 12 inclusive;and n is an integer from 1 to 3 inclusive; with the proviso that atleast one of R¹, R², R⁵, R⁶, R⁷, and R⁸ contains a PEG group.
 2. Acompound selected from the group consisting of

wherein each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of aliphatic,heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO₃, a PEGgroup P-L-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a (poly)ethylene glycol group where the(poly)ethylene glycol group is (CH₂CH₂O)_(s) where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P-L-Z, and a caboxamidegroup -L-CONH—P-L-Z; each of R⁷ and R⁸ is the same or different and isindependently selected from H, SO₃, a PEG group P-L-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and a(poly)ethylene glycol group where the (poly)ethylene glycol group is(CH₂CH₂O)_(s) where s is an integer from 3-6 inclusive, a sulfonamide—SO₂NH—P-L-Z, or a caboxamide —CONH—P-L-Z; where L is selected from thegroup consisting of a divalent linear (—(CH₂)_(o)—, o=0 to 15), crossed,or cyclic alkane group optionally substituted by at least one atomselected from the group consisting of oxygen, substituted nitrogen,and/or sulfur; where Z is selected from the group consisting of H, CH₃,alkyl, a heteroalkyl group, NH₂, —COO⁻, —COOH, —COSH, CO—NH—NH₂, —COF,—COCl, —COBr, —COI, —COO—Su (succinimidyl/sulfosuccinimidyl), —COO—STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′—CO—CH₂—I, —CONR′R″, —CONR′-biomolecule, —CONR′-L-COOH,—CONR′-L-COO—Su, —CONR′-L-COO—STP, —CONR′-L-COO-TFP, —CONR′-L-CONR″₂,—CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂, —CONR′-L-OH,—CONK′-L-O-phosphoramidite, —CONR′-L-CHO, —CONR′-L-maleimide, and—CONR′-L-NH—CO—CH₂—I; R′ and R″ is selected from the group consisting ofH, aliphatic, and heteroaliphatic, and the biomolecule is a protein,antibody, nucleotide, oligonucleotide, biotin, or hapten; X is selectedfrom the group consisting of —SH, —NH₂, —NH—NH₂, —F, —Cl, —Br, I, —NHS(hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O—STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH2-I, where R is —H, aliphatic, or heteroaliphatic; Kat isa number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s) needed tocompensate the negative charge brought by the cyanine; m is an integerfrom 0 to 5 inclusive; o is an integer from 0 to 12 inclusive; each ofR3 and R4 is the same or different and is independently —H, aliphatic,heteroaliphatic, or a PEG group P-L-Z where P is selected from anethylene glycol group, a diethylene glycol group, and a (poly)ethyleneglycol group where the (poly)ethylene glycol group is (CH₂CH₂O)_(s)where s is an integer from 3-6 inclusive; or R3 and R4 together form acyclic structure where R3 and R4 are joined using a divalent structuralelement selected from the group consisting of —(CH₂)_(q)—, —(CH₂)_(q)O(CH₂)_(q′)—, —(CH₂)_(q)S(CH₂)_(q′)—, —(CH₂)_(q)CH═CH—, —OCH═CH— whereeach of q and q′ is the same or different and is a integer from 2 to 6inclusive; and Y is selected from the group consisting of hydrogen,alkyl, sulfoalkyl, fluorine, chlorine, bromine, a PEG group P-L-Z whereP is selected from an ethylene glycol group, a diethylene glycol group,and a (poly)ethylene glycol group where the (poly)ethylene glycol groupis (CH₂CH₂O)_(s) where s is an integer from 3-6 inclusive, and anoxygen-containing group OR^(PM) where R^(PM) is selected from the groupconsisting of —H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cyclic alkyl,substituted or unsubstituted heterocyclic alkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl, wherethe group is optionally substituted one or more times with at least oneof hydroxyl, sulfo, carboxy, and/or amino; with the proviso that atleast one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ contains a PEG group. 3.A compound selected from the group consisting of

wherein each of R¹, R², R⁵, and R⁶ is the same or different and isindependently selected from the group consisting of aliphatic,heteroaliphatic, sulfoalkyl, heteroaliphatic with terminal SO₃, a PEGgroup P-L-Z where P is selected from an ethylene glycol group, adiethylene glycol group, and a (poly)ethylene glycol group where the(poly)ethylene glycol group is (CH₂CH₂O)_(s) where s is an integer from3-6 inclusive, a sulfonamide group -L-SO₂NH—P-L-Z, and a caboxamidegroup -L-CONH—P-L-Z; each of R⁷ and R⁸ is the same or different and isindependently selected from H, SO₃, a PEG group P-L-Z where P isselected from an ethylene glycol group, a diethylene glycol group, and a(poly)ethylene glycol group where the (poly)ethylene glycol group is(CH₂CH₂O)_(s) where s is an integer from 3-6 inclusive, a sulfonamidegroup —SO₂NH—P-L-Z, or a caboxamide group —CONH—P-L-Z; where L isselected from the group consisting of a divalent linear (—(CH₂)_(o)—,o=0 to 15), crossed, or cyclic alkane group that can be substituted byat least one atom selected from the group consisting of oxygen,substituted nitrogen, and/or sulfur; where Z is selected from the groupconsisting of H, CH₃, alkyl, heteroalkyl, NH₂, —COO⁻, —COOH, —COSH,CO—NH—NH₂, —COF, —COCl, —COBr, —COI, —COO—Su(succinimidyl/sulfo-succinimidyl), —COO—STP(4-sulfo-2,3,5,6-tetrafluorophenyl), —COO-TFP(2,3,5,6-tetrafluorophenyl), —COO-benzotriazole, —CO-benzotriazole,—CONR′—CO—CH₂—I, —CONR′R″, —CONR′-biomolecule, —CONR′-L-COOH,—CONR′-L-COO—Su, —CONR′-L-COO—STP, —CONR′-L-COO-TFP, —CONR′-L-CONR″₂,—CONR′-L-CO-biomolecule, —CONR′-L-CO—NH—NH₂, —CONR′-L-OH,—CONR′-L-O-phosphoramidite, —CONR′-L-CHO, —CONR′-L-maleimide, and—CONK-L-NH—CO—CH₂—I; R′ and R″ is selected from the group consisting of—H, aliphatic group, and heteroaliphatic group, and the biomolecule is aprotein, antibody, nucleotide, oligonucleotide, biotin, or hapten; X isselected from the group consisting of —SH, —NH₂, —NH—NH₂, —F, —Cl, —Br,I, —NHS (hydroxysuccinimidyl/sulfosuccinimidyl), —O-TFP(2,3,5,6-tetrafluorophenoxy), —O—STP(4-sulfo-2,3,5,6-tetrafluorophenoxy), —O-benzotriazole, -benzotriazole,—NR-L-OH, —NR-L-O-phosphoramidite, —NR-L-SH, —NR-L-NH₂, —NR-L-NH—NH₂,—NR-L-CO₂H, —NR-L-CO—NHS, —NR-L-CO-STP, —NR-L-CO-TFP,—NR-L-CO-benzotriazole, —NR-L-CHO, —NR-L-maleimide, and—NR-L-NH—CO—CH2-I, where R is —H, aliphatic, or heteroaliphatic; Kat isa number of Na⁺, K⁺, Ca²⁺, ammonia, or other cation(s) needed tocompensate the negative charge brought by the cyanine; m is an integerfrom 0 to 5 inclusive; p is an integer from 1 to 6 inclusive; each of R3and R4 is the same or different and is independently hydrogen, analiphatic group, a heteroaliphatic group, or a PEG group P-L-Z where Pis selected from an ethylene glycol group, a diethylene glycol group,and a (poly)ethylene glycol group where the (poly)ethylene glycol groupis (CH₂CH₂O)_(s) where s is an integer from 3-6 inclusive; or R3 and R4together form a cyclic structure where R3 and R4 are joined using adivalent structural element selected from the group consisting of—(CH₂)_(q)—, —(CH₂)_(q)O(CH₂)_(q′)—, —(CH₂)_(q)S(CH₂)_(q′)—,—(CH₂)_(q)CH═CH—, —OCH═CH— where each of q and q′ is the same ordifferent and is a integer from 2 to 6 inclusive; and Y is selected fromthe group consisting of —H, alkyl, sulfoalkyl, fluorine, chlorine,bromine, a PEG group P-L-Z where P is selected from an ethylene glycolgroup, a diethylene glycol group, and a (poly)ethylene glycol groupwhere the (poly)ethylene glycol group is (CH₂CH₂O)_(s) where s is aninteger from 3-6 inclusive, and an oxygen-containing group OR^(PM) whereR^(PM) is selected from the group consisting of hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cyclic alkyl, substituted or unsubstitutedheterocyclic alkyl, substituted or unsubstituted aryl, and substitutedor unsubstituted heteroaryl, where the group is optionally substitutedat least once with at least one of hydroxyl, sulfo, carboxy, and/oramino; with the proviso that at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷,and R⁸ contains a PEG group.
 4. The compound of claim 3 selected fromthe group consisting of


5. A method of labeling at least one biomolecule, the method comprisingproviding a composition comprising at least one excipient and thecompound of claim 2 in an effective concentration to label at least onebiomolecule under conditions sufficient for labeling the biomoleculewith the compound.
 6. The method of claim 5 wherein the biomolecule isselected from the group consisting of a protein, antibody, enzyme,nucleoside triphosphate, oligonucleotide, biotin, hapten, cofactor,lectin, antibody binding protein, carotenoid, carbohydrate, hormone,neurotransmitter, growth factors, toxin, biological cell, lipid,receptor binding drug, fluorescent proteins, organic polymer carriermaterial, inorganic carrier material, and combinations thereof.
 7. Amethod of labeling at least one biomolecule, the method comprisingproviding a composition comprising at least one excipient and thecompound of claim 3 in an effective concentration to label at least onebiomolecule under conditions sufficient for labeling the biomoleculewith the compound.
 8. The method of claim 7 wherein the biomolecule isselected from the group consisting of a protein, antibody, enzyme,nucleoside triphosphate, oligonucleotide, biotin, hapten, cofactor,lectin, antibody binding protein, carotenoid, carbohydrate, hormone,neurotransmitter, growth factors, toxin, biological cell, lipid,receptor binding drug, fluorescent proteins, organic polymer carriermaterial, inorganic carrier material, and combinations thereof.
 9. Themethod of claim 7 wherein the compound is V19-03005.
 10. A method ofdetecting at least one biomolecule, the method comprising providing acomposition comprising at least one excipient and the compound of claim2 in an effective concentration to detect at least one biomolecule underconditions sufficient for binding the compound to the biomolecule, anddetecting the biomolecule-bound compound.
 11. The method of claim 10wherein the biomolecule is selected from a protein, antibody, enzyme,nucleoside triphosphate, oligonucleotide, biotin, hapten, cofactor,lectin, antibody binding protein, carotenoid, carbohydrate, hormone,neurotransmitter, growth factors, toxin, biological cell, lipid,receptor binding drug, fluorescent proteins, organic polymer carriermaterial, inorganic carrier material, and combinations thereof.
 12. Themethod of claim 10 wherein the at least one biomolecule is detected inan assay selected from fluorescence microscopy, flow cytometry, in vivoimaging, immunoassay, hybridization, chromatographic assay,electrophoretic assay, microwell plate based assay, fluorescenceresonance energy transfer (FRET) system, bioluminescence reasonanceenergy transfer (BRET) system, high throughput screening, or microarray.13. The method of claim 10 wherein the biomolecule is detected by invivo imaging comprising providing the biomolecule-bound compound to atleast one of a biological sample, tissue, or organism, and detecting thebiomolecule within the at least one of a biological sample, tissue, ororganism.
 14. A method of detecting at least one biomolecule, the methodcomprising providing a composition comprising at least one excipient andthe compound of claim 3 in an effective concentration to detect at leastone biomolecule under conditions sufficient for binding the compound tothe biomolecule, and detecting the biomolecule-bound compound.
 15. Themethod of claim 14 wherein the biomolecule is selected from a protein,antibody, enzyme, nucleoside triphosphate, oligonucleotide, biotin,hapten, cofactor, lectin, antibody binding protein, carotenoid,carbohydrate, hormone, neurotransmitter, growth factors, toxin,biological cell, lipid, receptor binding drug, fluorescent proteins,organic polymer carrier material, inorganic carrier material, andcombinations thereof.
 16. The method of claim 14 wherein the at leastone biomolecule is detected in an assay selected from fluorescencemicroscopy, flow cytometry, in vivo imaging, immunoassay, hybridization,chromatographic assay, electrophoretic assay, microwell plate basedassay, fluorescence resonance energy transfer (FRET) system,bioluminescence reasonance energy transfer (BRET) system, highthroughput screening, or microarray.
 17. The method of claim 14 whereinthe biomolecule is detected by in vivo imaging comprising providing thebiomolecule-bound compound to at least one of a biological sample,tissue, or organism, and detecting the biomolecule within the at leastone of a biological sample, tissue, or organism.
 18. The method of claim14 wherein the compound is V19-03005.
 19. A kit for labeling and/ordetecting at least one biomolecule in a sample, the kit comprising thecompound of claim 2 and at least one excipient, and instructions for useof the compound to label and/or detect a biomolecule in a sample.
 20. Akit for labeling and/or detecting at least one biomolecule in a sample,the kit comprising the compound of claim 3 and at least one excipient,and instructions for use of the compound to label and/or detect abiomolecule in a sample.
 21. The kit of claim 20 wherein the compound isV19-03005.